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'1‘; 1 351% 2‘
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Michigan Scare
University

 

This is to certify that the
thesis entitled

REPRODUCTIVE PHYSIOLOGY OF MACACA FASCICULARIS
I. SEASONAL EFFECTS IN THE MALE
2. SEMEN PRESERVATION
3. LAPAROSCOPY
presented by

James P. Mahone

has been accepted towards fulfillment
of the requirements for

 

Ph.D. degree in _An_'Lma_|_I:Lusbandry-
Physiology

W fwA/WM

Major professor

Date October 3, I977

0-7 639

REPRODUCTIVE PHYSIOLOGY OF MACACA FASCICULARIS

 

l) SEASONAL EFFECTS IN THE MALE
2) SEMEN PRESERVATION

3) LAPAROSCOPY

BY

James Patrick Mahone

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Departments of Animal Husbandry and Physiology

1977

ABSTRACT .

REPRODUCTIVE PHYSIOLOGY OF MACACA FASCICULARIS
l) SEASONAL EFFECTS IN THE MALE
2) SEMEN PRESERVATION
3) LAPAROSCOPY

 

BY

James Patrick Mahone

These studies were conducted to determine the effect of
season on several reproductively important variables in
laboratory housed male cynomologus macaques. Additionally,
semen collection and preservation were investigated in the
male while the effect of extensive laparoscopy on reproduc-

tive performance was examined in female g. fascicularis.

 

Seasonal reproduction in laboratory housed adult male

fl. fascicularis was evaluated through examination of changes

 

in body weight, sperm concentration and testis volume over

a 13 lunar month period. No effect (p>’0.05) of season on
body weight or sperm concentration was observed. Testis
volume was significantly greater (p< 0.05) during the months
of July through early September than during October through
January (the times of greatest difference in mean testicular
volumes) as well as during a standardized comparison inter-
val with mean values during May through July being greater

than during October through January.

James Patrick Mahone

Semen was collected from males using a rectal probe for
electroejaculation. Power was supplied to the probe by a
square wave generator. Pulse width ranged from 1.8 to 7.0
msec, pulse frequency from 35 to 75 Hz with a 40 volt maxi-
mum output.

The effect of varying semen extender components and the
equilibration times of spermatozoa with glyceral prior to
pellet freezing was examined. No significant difference was
observed between motilities at extender egg yolk concentra-
tion of from 10 to 40%. Progressive motility was signifi-
cantly greater at pH's of 7.2 and 8.0 than at 5.8, 6.5 or
8.7 (p‘<0.05). Glycerol concentrations of 7 and 10% yielded
optimum motility after freezing. A one minute equilibration
of extender containing glycerol resulted in greater motility
after freezing than did equilibration with glycerol for 25
or 45 minutes (p<<0.05).

Thirty-six mating trials were conducted over an eleven
month period with sixteen female cynomologus macaques that
had been extensively laparoscoped. Five (31.3%) of these
females conceived. Two abortions from unknown causes
occurred at 112 and 123 days gestation to animals which had
been laparoscoped 43 and 54 times previously. Three females
that had been laparoscoped 38, 47 and 49 times prior to

breeding gave birth to live healthy infants.

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to my advisor,
Dr. W. Richard Dukelow, for his guidance, encouragement,
example and friendship during my graduate career. I thank
Drs. Harold Hafs, Robert Merkel, Ned Robinson, Duane Ullrey
and Dan Fox for their advice and counsel while participating
as members of my graduate committee.

No acknowledgements would be complete without recogni-
tion of Harry, John, Mac, Oscar, Al, Ernie and Archie who
contributed so much to this work.

The staff and graduate students at the Endocrine
Research Unit made work enjoyable through their help and
friendship. Special thanks go to Bonnie Cleeves for her
help particularly during the waning seconds of the prepara-
tion of this thesis.

Finally, I want to express my deepest gratitude to my
wife, Susan, and my children, Kristen and Eric, without
whose love, encouragement and sacrifice none of this would

have been possible.

 

ii

TABLE OF CONTENTS

Page

LIST OF TABLESOOOOOOOOOOO ......... OOOOOOOOOOOOOOOOOOO V

LIST OF FIGURES 0000000000 0.0000000000000000000000000. Vi

 

INTRODUCTION ....... 0.00.00.0000000000COOCOOOIOOOOOOC. l

l) SEASONAL REPRODUCTION IN THE LABORATORY HOUSED
MALE CYNOMOLOGUS MACAQUE, MACACA FASCICULARIS..... 3

 

 

Abstract....................................... 4
Introduction................................... 5
Materials and Methods.......................... 9
Results........................................ 11
Sperm Concentration......................... 11
Testis Volume............................... 14
Body Weight................................. 14
Discussion..................................... 19
References..................................... 25

2) SEMEN COLLECTION AND PRESERVATION IN MACACA
FASCICUI‘ARIS.I.0....C..00...OCOCOOOOCOOCOCCCOOOCOO 28

 

Abstract....................................... 29
Introduction................................... 30
Semen Collection............................ 30
Semen Preservation.......................... 32
Materials and Methods.......................... 36
Semen Collection............................ 36
Semen Preservation.......................... 37
Experiment l--Egg Yolk Concentration..... 38
EXperiment 2--pH Levels.................. 38
Experiment 3--Glycerol Concentrations.... 39

Experiment 4--Glycerol Equilibration Time
Before Freezing....................... 39

iii

TABLE OF CONTENTS--continued Page

Results ....................... . ..... .......... 40
Experiment l......... ..... _ ....... .......... 41
Experiment 2 ............ . ........... ....... 41
Experiment 3 ........... ... ..... ............ 46
Experiment 4............................... 46

Discussion. ........ ........................... 51

References.................................... 54

3) REPRODUCTIVE PERFORMANCE IN MACACA FASCICULARIS
FOLLOWING EXTENSIVE LAPAROSCOPY.................. 57

 

Abstract........................ ...... ........ 58
Introduction.. ....... ......................... 59
Materials and Methods......................... 61
Results..................... ..... ............. 63
Discussion and Conclusion... ...... ............ 63
References.................................... 66

SUMMARY AND CONCLUSIONS ...... . ........ .............. 68
PUBLICATIONS BY THE AUTHOROOOOOOCOOOOOOOOOO0.0.0.... 70

VITA.... ......... . .......... ........................ 71

iv

TABLE

LIST OF TABLES

Effect of Egg Yolk Concentration on the Motility
of Macaque Sperm After Freezing.................

Effect of Egg Yolk Concentration as a Percent of
Original Motility of Macaque Sperm After Freez-

lng ........ ......IOOOOOOOOO ..... ......OOOOOOOOOO

Effect of pH Level on the Motility of Macaque
Sperm After Freezing............................

Effect of pH Level as a Percent of Original
Motility of Macaque Sperm After Freezing........

Effect of Glycerol Concentration on the Motility
of Macaque Sperm After Freezing.................

Effect of Glycerol Concentration as a Percent of
Original Motility of Macaque Sperm After Freez-

lug-0.0.0.0...0.000.000.0000.........‘OOOOOOOOCOO

Effect of Glycerol Equilibration Time on the
Motility of Macaque Sperm After Freezing........

Effect of Glycerol Equilibration Time as a Per-
cent of Original Motility of Macaque Sperm After
FreeZingOOOOOOOO ...... O.......OOOOOIOOOOOOOOOOOO

Summary of Results of Mating Trials Conducted
Between May 1, 1975 and March 18, 1976..........

Page

42

43

44

45

47

48

49

SO

64

LIST OF FIGURES

FIGURE Page

1. Variations in sperm concentration over a 13
lunar month period in laboratory housed male,
Macaca fascicularis............................ 13

 

2. Variations in average testis volume over a
13 lunar month period in laboratory housed
male, Macaca fascicularis...................... l6

 

3. Variations in body weight over a 13 lunar month
period in laboratory housed male, Macaca
faSCiculariSII00............OOOOOCOOOOOCOCOOOOO 18

 

4. Mean values for chantes in sperm concentration,
average testis volume and body weight over a
13 lunar month period in laboratory housed
male, Macaca fascicularis...................... 21

 

vi

INTRODUCTION

Nonhuman primates are used extensively in biological
research providing the closest approximation to man for many
experimental investigations. Despite this need, economic
factors as well as the conservation of wild primate popula-
tions place a premium on the use of these animals for
research purposes. As a result, captive breeding colonies
may become a vital source of supply for future research sub-
jects.

The low fertility of captive nonhuman primates acts as
a major hurdle to the development of successful breeding
programs. One of the factors contributing to reduced fer-
tility is the seasonal effect on reproduction in both sexes
of many species. Previous studies have established the non-
seasonality of ovulation in the laboratory housed female
cynomologus macaque. Investigation was needed to determine
if this same condition exists in the male of that species.

The success of artificial insemination techniques using
frozen semen in domestic animals is indicative of its po-
tential value in a primate breeding colony. Application of
this technique would have the potential for obviating

seasonal effects on reproduction as well as allowing

extension of the breeding potential of superior males. No
live birth of a nonhuman primate from frozen semen has been
reported, indicating the need for research in this area.
Knowledge of the time of ovulation is essential for any
intensive breeding regimen. Laparoscopy provides the most
rapid and reliable method of ovarian observation in nonhuman
primates and as such is invaluable when artificial insemina-
tion or timed matings are used. The effect of repeated use
of this procedure on normal reproductive cyclicity and preg-
nancy was unknown at the time of initiation of these studies.
The objectives of these investigations were:
1. To quantify changes in reproductive variables in the male

Macaca fascicularis throughout the year.

 

2. To develOp a method for routine semen collection in

Macaca fascicularis.

 

3. To examine the effect of alteration of semen extender
components and the effect of short equilibration periods
of spermatozoa with glycerol on the motility of cynomo-
logus macaque sperm after freezing.

4. To evaluate the effect of laparoscopy on fertility in the

female Macaca fascicularis.

 

l) SEASONAL REPRODUCTION IN THE LABORATORY HOUSED
MALE CYNOMOLOGUS MACAQUE, MACACA FASCICULARIS

 

SEASONAL REPRODUCTION IN THE LABORATORY HOUSED
MALE CYNOMOLOGUS MACAQUE, MACACA FASCICULARIS

Abstract.

Changes in body weight, sperm concentration and testicu-
lar volume were monitored for a 13 lunar month period in

seven laboratory housed adult male M. fascicularis. No sig-

 

nificant change was noted in body weight or sperm concen-
tration between periods of maximal and minimal mean values
nor between periods standardized for comparison between the
three parameters. Testis volumes were significantly greater
(p<=0.05) during the months of July through early September
and during the standardized period of May through July than
during January through March and the standardized interval

October through January.

Introduction

 

A large number of mammals are seasonal breeders being
reproductively efficient for only brief periods during the
year. Among the environmental influences that may trigger
this phenomenon, photoperiod, temperature and humidity are
the most important.

Until recently nonhuman primates were not thought to
be seasonal breeders (Zuckerman, l93la,b). Recent evidence
however, has revealed a seasonal influence in many primate
species. Included in this list are ringtailed lemurs, Egmu£_

catla (van Horn, 1975); Macaca fuscata (Namura et al., 1971);

 

M. mulatta (Southwick et al., 1961 and 1965; Koford, 1965;

Prakash, 1958, 1960); M. radiata (Simonds, 1965), Presbytis

 

ventellus (Jay, 1965); Papio hamadryas (Kummer, 1968);

 

Cercopitheus kolbi (Omar and DeVos, 1971); M, sylvanus

 

(MacRoberts and MacRoberts, 1966), Saimiri sciureus (Dumond,

 

1968; Harrison and Dukelow, 1973) and M. fascicularis

 

(Dr. I. Berndston, 1976, personal communication).

While seasonal fluctuation in primate births have been
examined in feral groups, other criteria are more discrimi-
nating determinants of the male reproductive state. Among
these are gross changes in body morphology, testicular size
characteristics, semen characteristics, mating behavior and

hormonal influences.

Dumond and Hutchison (1967) observed increases in whole
body weight and heaviness of the upper torso now termed the
"fatted male" phenomenon in S. sciureus. This condition
persists under constant laboratory conditions (Clewe, 1969).
Androgen treatment of castrated male S. sciureus resulted in
increased body size, weight and physical appearance similar
to the condition found in intact animals during the mating
season (Nadler and Rosenblym, 1972).

Sade (1964) reported a seasonal fluctuation in testis
size in M. mulatta at Cayo Santiago, Puerto Rico. The great-
est length and width were observed during the warm wet
seasons during which the period of daylight was lessening.
This was in contrast to decreased testicular size during the
cool dry months of increasing photoperiod. The color of the
male sex skin was brightest during the season of greatest
testicular size and least intense during the season corre-
sponding to diminished testicular dimensions.

Rhesus monkeys demonstrate maximum testicular tubule
development during the breeding season (September through
November) with all spermatogenic and spermiogenic stages
present in high numbers (Conway and Sade, 1965). Degenera-
tive changes appeared starting in December and reached a
maximum from January through May. In June and July develop-

ment was reinitiated and progressed rapidly.

Zamboni et al. (1974), examined semen production in
male rhesus at Cayo Santiago. During the breeding season
semen production was normal in terms of volume, quality and
physical characteristics. Samples had a highly uniform
population of motile, structurally normal sperm. During the
nonbreeding season, males did not respond to electrostimula-
tion or produced only minimal quantities of ejaculate with
abnormal physical characteristics.

Drickamer (1974) theorized that the onset of the fall
mating behavior in rhesus is a result of interaction of many
factors. The onset of mating was observed to follow vegeta-
tion changes as a result of heavy rains in the late summer
(Vandenbergh and Vessey, 1968). Gordon et a1. (1976)
described c0pulatory activity as being restricted to a five
month period commencing in the fall of each year in rhesus
maintained in an outdoor compound in Lawrenceville, Georgia.

The seasonal pattern of mating behavior and testo-
sterone_rhythmicity observed in the wild, is maintained for
years following introduction of the male rhesus into the
laboratory environment (Robinson et al., 1975).

The rhesus monkey is a widely used primate research
model; however, this seasonal reproductive phenomenon is
much more marked in this species than is found in humans or
in some other nonhuman primate Species. Research using a

different, less reactive, primate would seem more appropriate.

Dang (1977) examined 275 menstrual cycles over a 3 year

period in 55 female M. fascicularis. These animals were

 

housed in a laboratory in Paris at_constant humidity and
temperature but under natural daylight. There was no
statistical difference in conception rate between four day-
light ratios examined (Short, 8-9 hours, increasing from
9—15 hours; long, 15—16 hours, decreasing from 16 to 8 hours).
No seasonal difference was observed in duration of menstrual
cycle length. Dukelow (1977) reported a slight seasonal
effect for cycle length, with longer cycles occurring during
the spring of the year and with slightly shorter cycles in
the winter months. No seasonal effect was noted in the

length of menstrual cycle flow. The M, fascicularis in this

 

study were observed for a five year period. Lighting was
controlled at a 12:12 hour 1ight:dark cycle with temperature
regulated between 21—26°C and relative humidity fluctuating
between 40-60% according to season. Both of these investi-
gations emphasize the difference in the seasonality of
reproduction in this species compared with the rhesus female.
No information is available on the persistence of sea-
sonal reproductive patterns in the laboratory housed male
cynomologus macaque. The objective of the present study was
to define the extent of seasonal reproduction in the labora-

tory housed male M. fascicularis. Three criteria were used

 

to evaluate the reproductive status of the males:

1. Concentration of Spermatozoa.
2. Testis volume.
3. Body weight.

Materials and Methods

 

This study was conducted from April, 1976 through March,

1977 with a colony of 7 adult male M. fascicularis at the

 

Endocrine Research Unit. All animals were adults and had
been in the colony for periods ranging from five months to
nine years at the time this study was initiated. Five of
the seven males were captive born. The macaques were indi-
vidually housed in 122 cm double unit modular cages in quar-
ters controlled at 21-26°C. Lighting was controlled on a
12:12 hour 1ight:dark cycle (0600-1800). The relative
humidity fluctuated between 40-60% according to season.
Females were individually housed in the same room. The
number of females present ranged from 2 to 28 over the 12
month period. All data were recorded on a lunar month (28
days) basis.

Animals were anesthetized with an intramuscular injec-
tion of Ketamine HCL (Vetalar, Parke Davis) at a dosage of
10 mg/kg body weight before sampling procedures. Every 28
days animals were anesthetized for three days in succession
between 1700 and 1900 hrs. Testicular measurements and body
weights were obtained during this interval. Body weights

for the three day period were averaged. Three testicular

10

measurements were taken with a micrometer on the length,
breadth and width of each testis on each of the three days.
Nine values were obtained for each 3 days interval and
averaged.

The testis volume was assumed to approximate that of
an ellipsoid. The volume was obtained according to the
formula for an ellipsoid, V = 4/3n(LXWXB). Both calculated
testis volumes were averaged, and the single value obtained
was statistically analyzed.

Semen was collected by electroejaculation (described in
the Materials and Methods section on Semen Collection and
Preservation) using a rectal probe. After incubation at 37°C
for 30 minutes, the liquid ejaculate was diluted (1:20) with
distilled water to stop all sperm motility. The spermatozoa
concentration was then determined with a hemocytometer. As
other experiments were in progress using the male macaques,
the first ejaculate taken after weighing and testis measure-
ments was used. This allowed a minimum of one week to
elapse between electroejaculation to eliminate any effect of
frequency of semen collection on sperm concentration.

All data for sperm concentration, body weight and testis
volume were analyzed using Scheffe's interval (Scheffe, 1953)
to contrast data selected after the experiment was conducted.
This procedure was used as no information is available on

when seasonal peaks are to be expected in the laboratory

ll

housed males. If drifting of seasonal reproductive peaks
or re-emergence of seasonal patterns occurred more restric-

tive analyses would be unable to detect them.

Results

Two separate statistical comparisons were made for each
factor examined (i.e., sperm concentration, mean testis
volume and mean body weight).

The first comparison involved contrasting mean values
for three consecutive months of highest values with the
corresponding three consecutive months during which the low-
est levels were observed. A second comparison was made
involving a standard non-breeding season (July through the
end of August) versus the pinnacle of the breeding season

of feral nonhuman primates (October through January).

Sperm Concentration

 

The results of fluctuations in sperm concentration for
all 7 males over the thirteen lunar month examination period
are presented in Figure l. The seasonal comparisons that
were statistically analyzed based on this data were:

I July (6-8), Aug. (3~5), Aug. (3l-Sept. 2)

Sept. (28-39(, Oct.vI26-28), Nov. (23-25)
II July (6—8), Aug. (3-5), Aug. 3l-Sept. 2
vs.

Oct. (26-28), Nov. (23-25), Dec. (21-23), Jan.
(19-21).

 

12

 

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14

There was no significant difference (p> 0.05) between sperm

concentrations for either of the contrasts made.

Testis Volume

 

Figure 2 reflects changes in testis volume over the
thirteen month period. Based on the mean values for each
month the following comparisons were made:

I Jul. (6-8), Aug. (3-5), Aug. 3l-Sept. 2

Jan. (19-21), FebYSIlS-l7), Mar. (15-17)
II Jul. (6-8), Aug. (3-5), Aug. 31-Sept. 2
Oct. (26-28), Nons(23-25), Dec. 21-23, Jan. (19-21)

Testis volume was significantly greater (p< 0.05) dur-
ing the months of July through the beginning of September
when compared to the interval January through March (repre-
senting the lowest testes volume attained. Furthermore,

this same difference was observed to exist (p< 0.05) when a

comparisons of the standard breeding seasons were made.

Body Weight

 

Figure 3 outlines variation in mean body weight over
the study. The periods during which high and low values
were compared as well as "standard" seasonal comparison were:

I May (11-13), June (8-10), July (6-8)
vs.
Oct. (26-28), Nov. (23-25), Dec. (21-23)

II July (6-8), Aug. (3-5), Aug. 31-Sept. 2
vs.
Oct. (26-28), Nov. (23-25), Dec. (21-23), Jan.
(19-21).

15

 

 

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19

Neither comparison for the months of high versus low body
weights or breeding versus nonbreeding season were statis-
tically different (p3>0.05).

Average values for all three criteria examined over the
thirteen 28 day intervals are shown in Figure 4. Since no
statistically significant change in either sperm concentra-
tion or body weight occurred, no attempts at correlating
either of these characteristics with each other or with

testis volume were made.

Discussion

 

The existence of an annual rhythm in sexual performance
and motivation, as distinguished from breeding, has been
well-documented in the laboratory housed male rhesus (M.
mulatta). This rhythmicity is extremely persistent in the
absence of photoperiod and temperature fluctuation in the
intact male as androgen rhythms continue under these condi-
tions (Michael and Zumpe, 1976; Michael and Bonsall, 1977).
Annual changes in behavioral performance in the castrate
male can occur in the absence of any major changes in plasma
androgen levels (Michael et al., 1975). Michael and Wilson
(1975) demonstrated that castrated males receiving exogenous
testosterone, and paired with ovarioectomized estrogen
treated females continued the same annual rhythm in mating

performance as did intact males, although the annual changes

20

 

 

 

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22

were less dramatic than for intact males.
The behavioral status of the rhesus is markedly differ-

ent than that of the laboratory housed male M. fascicularis.

 

Unpublished observations from our laboratory indicate that
male cynomologus macaques are sexually active and the
females receptive and capable of conception during every
month of the year.

Valerio (1969) has reported a very rapid decrease in

the annual reproductive cycle of M. fascicularis which re-

 

flects on both male reproductive efficiency as well as that
of the female.

Analysis of data from the present study demonstrates
that a definite seasonal effect on reproduction exists as
far as changes in testicular size are concerned. The impor-
tance of this effect to the overall reproductive efficiency
of the male is uncertain, particularly in light of the lack
of corresponding significant change in sperm concentration
or body weight for either high mean vs. low mean intervals
or during the "standard" breeding season. It is interesting

that the smallest testicular volume in M. fascicularis occurs

 

during the fall-winter months. This is the period during
which maximal volumes would normally be expected in the
rhesus. The most important parameter, sperm concentration,
shows no statistical variation. This, when considered with

lack of rhythmic changes in body size, would indicate a

23

stability of annual male reproductive characteristics not
exhibited by M, mulatta.

It is probable that any changes in reproductive patterns
would be reflected in hormonal variation.

Increases in androgen levels associated with reproduc—
tive function could potentially be influenced by factors
other than seasonal triggers. The effects of ejaculation

on testosterone levels in M. arctoides (Goldfoot et al.,

 

1975) and M. mulatta (Phoenix et al., 1977) have been
examined. In neither species were testosterone levels ele-
vated. Testosterone concentrations were monitored in these
experiments for several hours after ejaculation. This indi-
cates that stimuli other than mating would bring about
increases in testosterone during the breeding season.
Position in the primate social order appears to have
an extremely important influence on testosterone levels.
Rose et al. (1975) observed a fall in testosterone levels
in adult male rhesus following fighting defeat while the
assumption of alpha (highest social) status led to an in-
crease in testosterone levels. It was proposed that defest
produced a fall in testosterone with the lower androgen
concentrations decreasing the probability of aggressive
action on the part of the subject. This would reduce the
possibility for instigation of further combat and repeated

defeats.

24

A complete evaluation of persistence of seasonal repro-

ductive variation in M. fascicularis must include determina-

 

tion of total androgen or testosterone levels. Full clarifi-
cation of the environmental cues responsible for triggering
seasonal variation must include rigid control of humidity
as well as photoperiod and temperature.

Based on the present study it appears that an attenua-
tion of the variability of annual reproductive characteris-

tics occurs in the laboratory housed male M. fascicularis.

 

This species is, therefore, a much more suitable model for
reproductive studies related to the human than the more

commonly used M. mulatta.

25

References

 

Clewe, T. H. 1969. Observations on reproduction of squirrel
monkey in captivity. J. Reprod.-Fert. Suppl. 6:151-156.

Conway, C. H. and Sade, D. S. 1965. The seasonal spermato-
genic cycle in free ranging rhesus monkeys. Folia
Primat. 3:1-12.

Dang, D. C. 1977. Absence of seasonal variation in the
length of the menstrual cycle and the fertility of the
crab eating macaque (Macaca fascicularis) raised under
natural daylight ratio. Ann. Biol. anim. Bioch.
BiOphys. 17:1-7.

 

Drickamer, L. C. 1974. A ten-year summary of reproductive

data for free-ranging Macaca mulatta. Folia Primat.
21:61-80.

 

Dukelow, W. R. 1977. Ovulatory Cycle Characteristics in
Macaca fascicularis. J. Med. Primat. (In press).

 

Dumond, F. V. and Hutchinson, T. C. 1967. Squirrel monkey
reproduction: The Fatted male phenomenon and seasonal
Spermatogenesis. Science, 158:1467-1470.

Dumond, F. V. 1968. The squirrel monkey in a seminatural
environment. In: The Squirrel Monkey. L. A. Rosen-
blum and R. W. Cooper, eds., pp. 88-145. New York:
Academic.

 

Goldfoot, D. A., Slob, A. K., Scheffler, G., Robinson, J. A.,
Wiegand, S. J. and Cords, . 1975. Multiple
ejaculations during prolonged sexual tests and lack of
resultant serum testosterone increases in male stump-
tailed macaques (M, arctoides). Arch. Sex. Behav.
5:547-560.

 

Gordon, T. P., Rose, R. M., B-rnstein, I. S. 1976.
Seasonal rhythm in plasma testosterone levels in the
rhesus monkey (Macaca mulatta): A three year study.
Horm. Behav. 7:229-243.

 

Harrison, R. M., and W. R. Dukelow. . Seasonal adapta-
tion of laboratory maintained squirrel monkeys (Saimiri
sciureus). J. Med. Primatol. 2:277-283.

Jay, P. 1965. The common langur of North India: In:
Primate Behavior, I. Devore, ed., pp. 197-249.
New York: Holt, Rinehart and Winston.

 

26

Kirk, R. E. 1968. Experimental Design: Procedures for
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Puinshing Co., pp. 90-91.

 

 

Koford, C. B. 1965. Population dynamics of rhesus monkeys
in Cayo Santiago. In: Primate Behavior, I. DeVore,
ed., p. 160. New York: Holt, Rinehart and Winston.

 

Kummer, H. 1969. Social organization of hamadryas baboons.
Biol. Primat. 6:1-189.

MacRoberts, M. H. and MacRoberts, B. R. 1966. The annual
reproductive cycle of the Barbary Ape (Macaca sylvana)
in Gibraltar. Am. J. Phys. Anthrop. 25:299-304.

 

Michael, R. P. and Bonsall, R. W. 1977. A three year study
of an annual rhythm in plasma androgen levels in male
rhesus monkeys (Macaca mulatta) in a constant laboratory
environment. J. Reprod. Fertil. 49:129-131.

Michael, R. P. and Wilson, M. I. 1975. Mating seasonality
in castrated male rhesus monkeys. J. Reprod. Fertil.
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Michael. R. P. and Zumpe, D. 1976. Environmental and endo-
crine factors influencing annual changes in sexual
potency in primates. Psychoneuroendo. 1:303-313.

Michael, R. P., Zumpe, D., Plant, T. M. and Evans, R. G.
1975. Annual c hanges in sexual potency of captive
male rhesus monkeys. J. Reprod. Fertil. 45:169-172.

Nadler, R., Dand. L. A. Rosenblum. 1972. Hormonal regula-
tion of the "fatted" phenomenon in squirrel monkeys.
Anat. Rec. 173:181-188.

Nomura, T., Ohsawa, N., Tajima, Y., Tanaka, T., Kotera, S.,
Ando, A., and Nigi, H. 1971. Reproduction of Japanese
monkeys. In: Symposium on the use of non-human prim-
ates for research on problems of human reproduction.
World Health Organization, pp. 473-482.

Omar, A. and DeVos, A. 1971. The annual reproductive cycle
of an African monkey (Cercopithecus Kolbi Neuman).
Folia Primat. 16:206-215.

 

Phoenix, C. H., Dixson, A. F., and Resko, J. A. 1977.
Effects of ejaculation on levels of testosterone, corti-
sol and luteinizing hormone in peripheral plasma of
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27

Prakash, J. 1958. The breeding season of the rhesus monkey
Macaca mulatta. Zimmerman in Rajosthan. J. Bomb. Nat.
Hist. Soc. 55:154.

 

Prakash, J. 1960. Breeding of mammals in the Rajasthan
desert. J. Mamm. 41:368-369.

Robinson, J. A., Scheffler, G., Eisele, S. G. and Goy, R. W.
1975. Effects of age and season on sexual behavior and
plasma testosterone and dihydrotestosterone concentra-
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mulatta). Biol. Reprod. 13:203-210.

Rose, R. M., Bernstein, I. S. and Gordon, T. P. 1975.
Consequences of social conflict on plasma testosterone
levels in rhesus monkeys. Pschy. Med. 37:50-61.

Sade, D. S. 1964. Seasonal cycle in size of testes of free-
ranging Macaca mulatta. Folia. Primat. 2:171-180.

 

Simmonds, P. E. 1965. The bonnet macaque in South India.
In: Primate Behavior, I. Devore, ed., pp. 175-179.
New York: Holt, Rinehart and Winston.

 

Southwick, C. H., Berg, M. A., and Siddigi, M. R. 1961.
” A population survey of rhesus monkeys in Northern India.

II. Transportation routes and forest area. Ecology
42:699-715.

Southwick, C. H., Berg, M. A., and Siddigi, M. R. 1965.
Rhesus monkeys in Northern India. In: Primate Behavior,
I. DeVore, ed., p. 111. New York: Holt, Rinehart and
Winston.

 

Valerior, D. A., Palotta, A. J. and Courtney, C. D. 1969.
Experiences in large scale breeding of simians for
medical experiment action. Ann. N. Y. Acad. Sci. 162:
282-296.

Vandenbergh, J. G. and Vessey, S. 1968. Seasonal breeding
of free-ranging rhesus monkeys and related ecological
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Van Horn, R. N. 1975. Primate Breeding Season: Photo-
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Primatol. 24:203-220.

Zamboni, C., Conaway, C. H. and Van Pelt, . 1974.
Seasonal changes in production of semen in free-ranging
rhesus monkeys. Biol. Reprod. 11:251-267.

2) SEMEN COLLECTION AND PRESERVATION
IN MACACA FASCICULARIS

 

28

29

SEMAN COLLECTION AND PRESERVATION
IN MACACA FASCIAULARIS

 

Abstract

Semen was collected from adult male M. fascicularis

 

using a rectal probe for electroejaculation. Power was
supplied to the probe by a square wave generator. Pulse
width ranged from 1.8 to 7.0 msec, pulse frequency from 35
to 75 Hz with a maximum voltage output of 40 volts. The
effect of varying semen extender egg yolk concentration, pH,
glycerol concentration and equilibration times of sperm with
glycerol or sperm motility was examined. No significant
difference was observed between motilities at extender egg
yolk concentrations of from 10 to 40%. Progressive motility
was significantly greater at pH's of 7.2 and 8.0 than at
5.8, 6.5 and 8.7 (p<<0.05). Glycerol concentrations of 7
and 10% yielded optimum progressive motility after freezing.
A one minute equilibration of extender containing glycerol
resulted in greater sperm motility after freezing than did

equilibration with glycerol for 25 or 45 minutes.

30

Introduction

 

Nonhuman primates are widely used in biomedical
research because of their physiological and anatomical simi-
larity to humans. Animals used as research subjects often
have no prior health records or information on their genetic
composition.

Many benefits could be derived from a successful
semen collection and preservation program to be utilized
with artificial insemination for increasing the production
of nonhuman primates in captivity. Semen from males selected
for possession of superior characteristics of high heritabil-
ity or for possession of traits valuable for the study of
inherited defects could be disseminated throughout the
research community. Seasonal declines in sperm production
could be obviated through successful semen preservation and
insemination techniques. Reduction in the total number of
males required to be maintained for breeding purposes would

be possible.

Semen Collection

 

Theoretically a wide variety of semen collection tech-
niques could potentially be used with nonhuman primates.
The artificial vagina is commonly used with domestic live—
stock. Attempts to use this technique with nonhuman primates
have yielded little success (D. D. Hoskins, personal communi-

cation). Roussel and Austin (1968) reported little success

31

in collecting semen from nonhuman primates by masturbation
or through stimulation with a massage vibrator. Semen has
been collected from the vaginas of naturally mated female
cynomologus macaques (Cho and Honjo, 1973). There are
several disadvantages to this procedure. First some of the
semen may be diluted and contaminated by vaginal secretions.
Another factor to be considered is the adverse effect of

the female tract environment on sperm. Finally, the use of
this procedure necessitates the use of additional receptive
females to be maintained for collection.

Electroejaculation is the technique most commonly used
for semen collection in nonhuman primates. With smaller
monkeys this technique can be successfully employed without
anesthesia (Kuehl and Dukelow, 1974). Mastroianni and
Manson (1963) have reported successful recovery of semen
following electroejaculation of unanesthetized macaques with
penile electrodes.

The more common method involves electroejaculation of
anesthetized animals with rectal electrodes. Rectal probes
have been effectively employed in a wide variety of nonhuman
primates (Fussel et al., 1967; Roussel and Austin, 1968;
Kraemer and Vera Cruz, 1969). Warner et a1. (1974) investi-
gated different current flow patterns and obtained optimum
collection using a circular pattern provided by longitudinal

electrodes.

32

The ejaculate of the nonhuman primate is generally
characterized by formation of a coagulum or plug that may be
accompanied by a liquid fraction. Different enzymatic agents
have been used to reduce or prevent coagulum formation and
to increase the number of spermatozoa in the liquid fraction
(Hoskins and Patterson, 1967; Van Pelt and Keyser, 1970;

Bush et al., 1975). The disadvantage of this approach lies
in the unknown effects of the various enzymes on sperm

fertility.

Semen Preservation

 

The discovery of Polge et a1. (1949) that glycerol pro-
tects spermatozoa during the freezing process has led to the
extensive use of frozen semen in domestic and exotic animal
breeding as well as in humans. The success of freeze preser-
vation is based on three factors: 1) the critical tempera-
ture ranges through which the spermatozoa pass before
reaching storage temperature; 2) the rate at which the semen
is cooled, and 3) the cryoprotective agent in which the
semen is extended (Ackerman and Behrman, 1968).

As spermatozoa are cooled they are subjected to cold
shock. This occurs as the semen is cooled from room tempera-
ture to about +5°C and is characterized by a rapid and perma-
nent loss of motility, decreased rate of metabolism and
decreased fertilizing capacity. Cold shock appears to in-

crease the permeability of the sperm cell (Mann, 1949).

33

The latent heat of crystallization is reached at from
+5°C to —5°C and is associated with initial ice crystal
formation. The sperm are subjected to a sharp temperature
increase at this point. Further crystallization occurs
down to -15°C.

The zone of recrystallization falls between -15°C and
-79°C. It is this range that the growth and reunion of ice
crystals into larger complexes occurs. Under any storage
system there is wide enough fluctuation in temperature to
allow for recrystallization. Once semen has been preserved,
an assessment of the efficiency of the technique used is
essential.

The absolute criterion for evaluation of successful
semen preservation is the attainment of pregnancy. This is
difficult in nonhuman primate research colonies where rela-
tively few animals may be available for insemination. Other
biochemical and morphological measures have been used in
situations where indirect evaluation of sperm activity is
required.

Nigrosin-eosin (live-dead) staining, in which dead
spermatozoa take up the stain, has been used to evaluate
sperm viability. Mittal (1975) has recently reviewed this
area. The efficiency of live-dead staining is usually com-

pared with motility for assessing semen quality.

34

Assay techniques for sperm enzymes have been employed
in a variety of different situations based on the premise
that as the sperm cell is frozen its cellular membranes are
damaged, resulting in the release of cellular material
(DeReuck and Knight, 1964; Brown et al., 1971; Stambaugh and
Buckley, 1968; Zaneveld et al., 1972; Foulkes and Watson,
1975; Schill, 1975). Morphological changes in the acrosome
during freezing also show promise as a predictor of potential
fertility (Saacke and White, 1972; Pursel et al., 1972).

The most rapid and reliable method of assessing the
viability of spermatozoa is progressive or forward motility.
The correlation with fertility is low.

Traditional attempts to successfully freeze nonhuman
primate semen have relied on techniques developed for use in
domestic animals. Equilibration for periods up to several
hours with or without glycerol at 4-5°C, followed by con-
trolled cooling to storage temperature or rapid pellet freez-
ing on dry ice have all been successful.

Good motility has been obtained after freezing in ex-
tenders containing egg yolk, glycerol, citrate and phosphate
buffers, following incubation at 37°C for 30 minutes in

Macaca mulatta (Leverage et al., 1972). Roussel and Austin

 

(1967) achieved good past thaw motility in an extender con-
sisting of egg yolk, sodium glutamate buffer and 14% glycerol.

Kraemer and Vera Cruz (1969) utilized this same extender,

35

plus penicillin and streptomyocin, to successfully freeze
Papio §E semen.
An extender consisting of glucose, lactose, raffinose

and 5% glycerol has been used to pellet freeze Macaca fasci-

 

cularis semen. This extender was maintained at a pH of 7.2
with tris buffer. Dennis et a1. (1976) have used a two step
equilibration period with extender containing 4% glycerol,
egg yolk and lactose to pellet freeze squirrel monkey semen.

The basic extender components usually include egg yolk
and glycerol. Lactose has been demonstrated to be a non-
penetrating cryoprotectant and is highly beneficial in any
rapid freezing technique (Rowe, 1966). pH level is normally
maintained in the physiological range. The discovery that
egg yolk has a beneficial effect by diminishing cold shock
(Phillips, 1939) has led to its widespread use in semen
extenders. Pace and Graham (1974) have demonstrated that
semen extenders containing 20% egg yolk, in the absence of
glycerol, protected the motility of bull sperm during the
freezing process. Berndston and Foote (1972) have demon-
strated that exposure of bull semen to glycerol for as short
as ten seconds is sufficient for successfully freezing,
indicating that glycerol may produce its effect very rapidly.

The objectives of the present study were:

1) To evaluate the efficacy of electroejaculation for

semen collection in M. fascicularis.

 

36

2) To examine the effect of egg yolk concentrate on the
viability of sperm when frozen.

3) To determine the effect of varying pH levels on the
viability of sperm subjected to freezing.

4) To determine if glycerol is required for the success-
ful pellet freezing of semen and at what levels.

5) To determine if a short equilibration with glycerol
prior to pellet freezing is as effective as longer equili-

bration periods.

Materials and Methods

 

Semen Collection

 

A rectal probe designed after Healy and Sadlier (1966)
was used for electroejaculation. Power was supplied to the
probe by a square wave generator. Pulse width ranged from
1.8 to 7.0 msec, pulse frequency 35 to 75 Hz and maximum
voltage output was 40 volts.

A sling was used to support the animal during semen
collection (Roussel and Austin, 1968).

Seven adult M. fascicularis males were used to develop

 

the semen collection technique. The animals were individual-
ly housed and received a commercially prepared monkey diet
(Wayne; Allied Mills Inc.) and water ad libitum. Following

an intramuscular injection (10 mg/kg body weight) of

37

Ketamine-HCL (Vetalar, Parke-Davis) to induce anesthesia,
the monkey was positioned in the support sling. Its feet
were secured to the rear posts of the frame and the rectal
probe inserted so that the last brass ring was just inside
the anal sphincter.

Stimulation was accomplished by gradually increasing
the voltage in an on-off pattern lasting from 2-5 seconds.
The pattern was maintained until ejaculation occurred.

Semen was collected in a 3 ml plastic vial and placed in an
incubator at 37°C for 30 minutes. After incubation, the
semen volume was measured and the sperm concentration deter-
mined with a hemocytometer. The percent progressive and
total motility was determined after dilution (1:20) in
physiological saline warmed to 37°C. At least 100 sperm
were counted and evaluated for motility. Progressive motil-
ity was defined as rapid forward motion of individual sperm
cells while total or raw motility was comprised of total

motile sperm, regardless of direction or Speed of movement.

Semen Preservation

 

A single extender was used for the semen freezing ex-
periments. The basic extender formula consisted of 4%
glycerol, 20% fresh egg yolk, and 11% lactose in distilled
water with individual variations depending on the variable
to be tested. The pH was adjusted, dependent on experiment,

with 4% tris buffer.

38

Following collection and equilibration of the semen at
37°C, the whole ejaculate was maintained at room temperature
for 10 minutes while sperm concentration, progressive and
total motilities were determined. The ejaculate was split
into 10 ul aliquots and at the end of equilibration at room
temperature, 45 ul of semen extended was added at 4°C. The
extended semen was then equilibrated at 4°C for 20 minutes.
An additional 90 pl of extender was added and equilibrated
with the semen for 25 minutes before pellet freezing on dry
ice.

After freezing, the semen was incubated at 37°C for 15
minutes. The percent of original progressive and total

motility was then determined.

Experiment 1--Egg Yolk Concentration

 

The extender used was composed of 4% glycerol, 11% lac-
tose and was buffered to a pH of 7.2 with 4% tris buffer.
Egg yolk concentration was 10, 20, 30 or 40%. Ejaculates
were collected from four males. Following collection, the
semen was equilibrated with extender and pellet frozen as
previously described. The data were analyzed using Dunnet's

t-test.

Experiment 2-epH Levels

 

The extender consisted of 4% glycerol, 11% lactose, 20%

egg yolk. It was buffered to a pH of 6.5, 7.2, 8.0 or 8.7

39

with 4% tris buffer. The pH of unbuffered extender was left
at 6.0. Four separate semen collections were made from each
of four males. Semen was equilibrated with extender and
pellet frozen on dry ice. Since a wide pH range has been
used to freeze primate semen, Scheffe's interval was used to

analyze the data collected.

Experiment 3--Glycerol Concentrations

 

An extender was formulated with 20% egg yolk, 11%
lactose and adjusted to a pH of 7.2 following addition of
glycerol. Glycerol levels used were 0, 4, 7, 10 and 20%.
Semen from three males was obtained on each of four days.
The semen was extended and frozen in the same manner as
described for experiments 1 and 2. The data were analyzed
using orthogonal f—tests for single degree of freedom con-
trasts. 7

Experiment 4—-Glycerol Equilibration
Time Before Freezing

 

 

An extender consisting of 20% egg yolk, 7% glycerol,
11% lactose and buffered to a pH of 7.2 with 4% tris buffer
was formulated. Equilibration times of 1, 25 or 45 minutes
with glycerol prior to freezing were examined for their
effect on post thaw motility.

Four males were collected on each of four days.

The protocol used for equilibration with extender dif-

fered from that used in the first three experiments.

4O

Semen equilibrated with glycerol for l or 25 minutes was
first placed in extender containing no glycerol for 20 min-
utes. At the end of this period, extender containing 14%
glycerol (to bring the final extender concentration to 7%)
was added and equilibrated at 4°C for l or 25 minutes before
pellet freezing on dry ice. The 45 minute treatment group
was handled in the same way as in the first three experi-
ments. Specifically, extender containing 7% glycerol was
added during both equilibration periods of 20 and 25 min-
utes. In all 3 treatment groups, the final extender compo-
sition was 20% egg yolk, 11% lactose and 7% glycerol at a
pH of 7.2. Orthogonal f—tests for single degree of freedom

contrasts were used to analyze the data.

Results

Semen was collected from seven male cynomologus
macaques using a rectal probe for electroejaculation. Of
410 trials, 407 resulted in successful collection of ejacu-
lates. The three unsuccessful attempts were a result of
stimulator malfunction.

Stimulation with the rectal probe for from 2 to 10 min-
utes resulted in good recovery of ejaculate volume. The
volume of the liquid parts of the sample following incuba-

tion at 37°C for 30 minutes, ranged from 10 ul to 1.5 m1.

41

Sperm concentration varied from 0 to 5 X log/ml with progres-
sive motility values from 0 to 97%.

Four males that had consistently yielded high concentra-
tion (in excess of 20 X 106/m1) following electroejaculation
were selected for use in subsequent semen freezing experi-

ments.

Experiment 1

Mean values for collections from each male, reflecting
the effect of egg yolk concentration on the sperm motility
after freezing, are shown in Table 1. The effect of this
treatment, expressed as a percent of original progressive
and original total motility, is depicted in Table 2. There
was no statistical difference (p=>0.05) between the percent
of original progressive or total motility for any of the egg

yolk concentrations examined.

Experiment 2

 

The effect of extenders buffered to varying pH's is
shown in Table 3. Motility is expressed as percent original
progressive and total motility in Table 4. Progressive
motility was significantly greater at pHs of 7.2 and 8.0
than at 5.8, 6.5, or 8.7. Total motility was significantly
greater (P<<0.05) at pH 8.0 when contrasted with that at
pH 8.7 but not between 8.0 and any of the other treatment

groups examined (p:>0.05).

42

 

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43

 

 

 

 

 

Table 2. Effect of Egg Yolk Concentration as a Percent of
Original Motility of Macaque Sperm After Freezing
Male Egg Yolk Concentration
Number 10% 20% 30% 40%
Prpgressive Motility
l 67 7O 57 97
2 O 15 13 31
3 27 51 57 67
4 22 28 100 44
i + SE 29:18.9 41:23.1 57:32.3 60:32.4

bWNH

XI
+

SE

Statistical Analysis for Progressive Motility

10% vs. 20% (p>-0.05)
10% vs. 30% (p:>0.05)
10% vs. 40% (p> 0.05)

Total Motility

 

100 100 71 97
31 27 25 67
37 59 74 85
29 74 100 68
49:28.7 65:35.1 68:36.4 79:40.1

Statistical Analysis for Total Motility

10% vs. 20% (p>'0.05)
10% VS. 30% (p>'0.05)
10% VS. 40% (P3>0.05)

 

 

44

 

 

 

 

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45

 

 

 

 

 

 

 

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mHmsHmca HmoHumHumum
o.HHm.e a.mun.om m.ank.mH m.mnm.e m.mum.m GH mm H m
k.Hmm.m m.mHmm.am m.OHmm.om o.mmm.a k.emH.m a e
m.mwm.m m.~HHm.mv m.~Hm.eH m.HHm.H o.oHo.o a m
m.mnn.e 8.0 HH.mH H.0Ho.~m m.enm.VH m.mHm.m a m
o.o+o.o m.mH+m.Hm e.m+m.m e.m+m.s m.H+G.m a H
NHHHHHOE o>HmmmHmmHm
k.m o.m N.h m.m o.m 2c. umnssz
Ho>mq mm mHmz
mcHnmmHm young
Eummm mswmomz mo quHHuoz HMCHmHHO mo unwoumm 6 mm Hm>mq mm mo uommmm .v mange

46

Experiment 3

 

Sperm motilities after equilibration in extenders con-
taining 0, 4, 7, 10 and 20% glycerol are given in Table 5.
The percent of original motility after freezing is depicted
in Table 6. Progressive motilities in extenders containing
glycerol at 7 and 10% concentrations were significantly
greater (p 0.05) than at 4%. There was no significant dif-
ference (p 0.05) between progressive motilities for
extenders containing 7 and 10% glycerol concentrations nor
between total motilities at 7 and 10% glycerol concentra-
tions. Total motility was greater at 7 and 10% glycerol than
at 4 and 20% (p 0.05). Extender containing no glycerol
resulted in no progressive or total motility and was excluded

from the statistical analysis.

Experiment 4

 

Table 7 shows the effect on motility of time of glycerol
equilibration prior to pellet freezing. The effect of equi-
libration with glycerol for l, 25 or 45 minutes prior to
pellet freezing on the percent of original progressive and
total motility is demonstrated in Table 8. Progressive
motility was significantly greater (p 0.05) after one minute
equilibration than following 25 or 45 minutes of equilibra-
tion with glycerol. No significant differences (p 0.05) was
observed between progressive motilities at 25 or 45% nor
between total motilities for any of the equilibration times

examined.

47

 

 

 

 

 

 

 

o.m+H.m e.mua.om m.aHH.om m.wwm.m~ o.oHo.o H.8Hm.mm NH mm+ m

a.mmm.a m.MHmo.ma 8.HHmH.oe o.mmo.mm o.omo.o v.emm.mm a a

G.HHG.4 m.~HG.mN m.HHa.mm m.mHo.om o.oHo.o s.mnm.ke a m

o.H+o.H m.v+m.~m m.v+~.mH m.H+v.e o.o+o.o a.m+a.mm a H

HHHHHuoz Hmuoe quuuma

N.oHN.o m.mHa.m m.NHH.a m.HHm.m o.oHo.o 8.0Hm.me «H mm H m

o.omo.o n.0mm.HH m.amm.OH m.Hmm.H o.omo.o o.mmm.vm a a

0.0Hm.o “.mHO.HH N.mHo.m o.mHH.m c.0Ho.o m.mue.mm a m

o.o+o.o m.H+H.e m.H+e.m m.o+H.H c.o+o.o m.m+a.m~ a H

.NuHHHuoz 0>HmWWMmoum unmoumm

mom 20H we I. as so maHecmuxm mnemmm c Honaaz

mm + M COHumuucmocou Honmomao muHHHuoz HmchHHO mam:
mcHNmmnm
Hound Eummm mnemomz mo muHHHHoz may no coflumuucmocou Houmomaw mo uommmm .m oHQma

48

 

.mo.o Va. won .38 .> 30H .35

.35 Am.

woa .> we

mHmaHmcd HMOHumHumum

 

 

 

 

 

 

«.mum.k m.MHum.mv «.mHH~.ms m.HHHH.mm o.o+o.o NH mm + m
o.HMo.mH H.5HmH.oe o.aHmm.mm H.8Hmo.~m o.omo.o a a
o.mne.m m.mHm.Hm G.Hnm.ev H.mH~.oe o.owo.o a m
m.H+o.m H.e+e.mm a.e+m.mm e.m+H.- o.o+o.o a H
HuHHHuoz Hmuoe
.mo.o Va. HOH .we .> we
Hmo.oxns 30H .> we
mHmmHmcd HMUHumHumum
o.ouo.o N.GHH.mH H.8Ho.mH o.mHv.H o.ono.o NH mm H m
o.omo.o o.HHmm.aH H.HHmm.kH v.4mH.m o.omo.o H e
o.oHo.o m.owm.eH m.mHN.o~ H.mHm.m o.oHo.o a N
o.o+o.o m.m+m.aH n.m+H.mH H.H+m.k o.o+o.o a H
NHHHHHOZ m>HmmmHmmHm
wow 30H we we so a umnaaz
coHumuucmocou Honmomau mHmz
wcflummum Hmuwm Eummm mswmomz
mo >uHHHuoz HmchHHO mo unmouom m we :OHumHucmocou Honmomaw mo pommmm .m magma

49

 

 

N.muo.mN H.NH0.NN H.4Hm.mm m.¢Hm.Nm mH mm + m
H.mmn.NN k.mmH.om 4.8mm.aN v.8HMN.Nv a a
e.mwm.eN «.mna.mm m.enm.ea N.mHo.VN a N
o.kna.km «.mwm.Ha 8.8Hm.Nm o.HHe.eN a N
m.m+m.NN e.m+m.NN m.m+m.eN m.a+m.om a H
HwHHHHoz Hmuoe unmouma
a.HHH.a a.HHN.m n.NHq.mH H.6HN.H8 mH mm H m
m.Hmm.H e.Nma.n m.amH.NH m.mHmN.mN a e
N.Nws.sH 0.8Hm.mH 0.8Hm.mN 0.8Hm.ka e N
H.mHN.NH H.8Hm.v o.eum.aH o.mHH.Hm a N
o.m+a.e N.a+m.m m.N+H.OH k.m+m.om a H

 

muHHHuoz o>HmmmHmonm unmoumm

 

mchcopxm muommm c Honasz
muHHHuoz HmchHuo 6H8:

mmuscHE me mmusnme mm ouscmm H
mEHB coHumHQHHHnwm HonoomHo

 

 

 

mcfluomum Hound
Eummm mdqmomz mo muHHHuoz ecu co mEHB coHumnnflHHsvm Houmomao mo uommmm .n wanes

50

Table 8. Effect of Glycerol Equilibration Time as a Percent
of Original Motility of Macaque Sperm After Freez-

 

 

 

 

 

 

ing
Male Glycerol Equilibration Time
Number n 1 Minute 25 Minutes 45 Minutes
Percent Progressive Motility
l 4 36.5:10.7 29.2:16.4 28.7:11.6
2 4 35.8:12.4 9.6:9.l 24.0:10.4
3 4 49.2:4.9 27.5:5.1 30.7:2.4
4 4 46.7:22.2 33.7:18.0 5.8:5.3
X : SE 16 42.0:11.4 25.0:8.4 22.3:6.6
Statistical Analysis
1 min2>25 min, 45 min (p‘<0.05)
25 min v. 45 min (p2>0.05)
Pegcent Total Motility
1 4 39.8:ll.2 38.2:15.5 38.7:3.6
2 4 62.8:5.7 49.2:5.8 45.7:9.4
3 4 64.8:8.2 55.4:9.7 37.9:6.9
4 4 70.1:13.6 72.1:16.4 60.3:24.3
Z : SE 16 59.4:14.3 53.7:13.3 45.6:12.1

Statistical Analysis

1 min v. 25 min, 45 min (p>>0.05)
25 min v. 45 min (p> 0.05)

 

51

Discussion

 

The semen collection trials demonstrated that electro-
ejaculation is a rapid and reliable method for obtaining

spermatozoa from M. fascicularis. The stress associated

 

with extensive anesthesia and electroejaculation is minimal
as is evidenced by continued good health and weight main-
tenance during these experiments. In View of these results,
rectal electroejaculation is the method of choice for semen
collection in this nonhuman primate.

The results of Experiment 1 indicate that a wide range
of egg yolk concentrations are effective as cryoprotectants
in the semen preservation process. Virtually all semen
extenders contain egg yolk as a constituent, but no addi-
tional benefit appears to be derived from increasing the

concentration above 20% in M. fascicularis.

 

The results of the second experiment demonstrate that
extenders in the pH range of 7.2 to 8.0 provide the better
post thaw motility. It is surprising that pH 8.0 would be
so effective, since extenders used previously for nonhuman
primate semen preservation have normally been adjusted to pH
7.2 (Cho and Honjo, 1973) or left at pH 6.0 (Denis et al.,
1974). Meizel and Mukerji (1975) have reported the greatest
activation of proacrosin to its active form acrosin is at pH
8.0. Acrosin is involved in the penetration of the zona

pellucida by the sperm. The greater motility obtained in

52

the pH range 7.2-8.0 may be related to this phenomenon in
that the higher pH results in increased metabolic activity
associated with motility.

The effects of glycerol levels as a cryoprotectant for
macaque Sperm during the freezing process are very straight
forward. The data Show a definite requirement for glycerol
as no motility was obtained after freezing when glycerol was
omitted from the extender. Glycerol levels similar to those
used with human semen (7—10%) were more effective than those
used for the squirrel monkey (Denis et al., 1974). The high
glycerol level (20%) was detrimental to spermatozoa probably
due to osmotic effects of the high concentration of the
solution.

The final investigation (Experiment 4) demonstrated
that, while glycerol is a required extender constituent for
freeze preservation, a very Short equilibration time with
glycerol prior to freezing results in maximum motility after
freezing. Berndston and Foote (1972) working with bull
semen, have suggested that glycerol has an immediate dehydra-
tion effect on sperm cells and that rapid freezing decreases
the effect of intracellular ice crystal formation and avoids
the effect of high osmotic pressure. An alternate hypothesis
is that glycerol may act as a sperm membrane stabilizer to
protect the cell during freezing but that longer incubation

may exert an osmotic influence resulting in cell damage.

53

Both the dehydration and membrane stabilization effect may
act simultaneously to protect the individual Sperm.

While insemination with fresh or extended seman has
been successful, no nonhuman primate inseminated with frozen
semen has given birth to live young. Leverage et a1. (1972)
artificially inseminated M. mulatta with frozen semen. Of
48 inseminations, only one resulted in pregnancy. This con-
ceptus was aborted at 40 days of gestation. Cho et a1.'
(1975) have obtained 8 successful pregnancies following 13
artificial inseminations in 8 animals with frozen semen.
These pregnancies resulted in abortion 6-8 weeks after con-
ception.

The result of the present study indicates the value of
examining different aspects of extender components and
freezing techniques. Future studies Should examine all
variables necessary to promote optimum motility in lieu of
making direct application of successful methods in one
species to nonhuman primates. .These studies must eventually
include insemination trials to assess fertility of the

frozen semen.

54

References

Akerman, D. R. and S. J. Behrman. 1975. Artificial insemi-
nation and preservation of human semen. S. J. Behrman
and R. W. Kistner, editors. In: Progress in Infertil-
£_y, 2nd ed., 765-778. Boston: Little, Brown and
Company.

 

Berndston, W. E. and R. H. Foote. 1972. The freezability
of spermatozoa after minimal pre-freezing exposure to
glycerol or lactose. Cryobiol. 9:57-60.

Brown, K. I., B. G. Crabo, E. F. Graham and M. M. Pace.
1971. Some factors effecting loss of intracellular
enzymes from spermatozoa. Cryobiol. 8:220-224.

Bush, D. E., L. H. Russel, Jr., A. I. Flowers and A. M.
Sorenson, Jr. 1975. Semen evaluation in capuchin
monkeys. Lab. Anim. Sci. 25:588-593.

Cho, F. and S. Honjo. 1973. A simplified method for col-
lecting and preserving cynomologus macaque semen.
Japan. Med. Sci. and Biol. 26:261-268.

Cho, F., S. Honjo, and T. Makita. 1974. Fertility of fro-
zen preserved spermatozoa of cynomologus monkeys.
Cont. Primat. 5th Intl. Cong. Primat. (Karger, Basel),
pp. 125-133.

Denis, L. T., A. N. Poindexter, M. B. Ritter, S. W. J.
Seager and R. L. Deter. 1976. Freeze preservation of
squirrel monkey Sperm for use in timed fertilization
studies. Fertil. Steril. 27:723-729.

DeReuch, A. V. S. and J. Knight Eds. 1964. CIBA Foundation
symposium on cellular injury, pp. 314-317. Ciba Founda-
tion, London, England.

Foulkes, J. A. and P. A. Watson. 1975. Hyaluronidase
activity in seminal plasma as a method of assessing
bull Sperm integrity. J. Reprod. Fert. 43:349-353.

Fussel, E. N., J. D. Roussel, and C. R. Austin, 1967.
Use of the rectal probe method for electrical ejacula-

tion of apes, monkeys and a prosimian. Lab. Anim. Care
17:528-530.

55

Healy, P. and R. M. F. S. Sadlier. 1966. The construction
of rectal electrodes for electroejaculation. J. Reprod.
Fert. 11:299-301.

Hoskins, D. D. and D. L. Patterson. 1967. Prevention of
coagulum formation with recovery of motile spermatozoa
from rhesus monkey semen. J. Reprod. Fert. 13:337-340.

Kirk, R. E. 1968. Experimental Design: Procedures for
Behavioral Sciences. Belmont, California: Wadsworth
Publishing Co., pp. 90-91.

Kraemer, D. E. and N. C. Vera Cruz. 1969. Collection,
gross characteristics and freezing of baboon semen.
J. Reprod. Fert. 20:345-348.

Kuehl, T. J. and W. R. Dukelow, 1974. A restraint device
for electroejaculation of squirrel monkeys (Saimiri
sciureus), Lab. Anim. Sci. 24:364-366.

Leverage, W. P., D. A. Valerio, A. P. Schultz, E. Kingsbury
and C. Dorey. 1972. Comparative study on the freeze
preservation of spermatozoa. Primate, bovine and human.
Lab. Anim. Sci. 22:882-889.

Mastroianni, L. A. and w. A. Manson, Jr. 1963. Collection
of monkey semen by electroejaculation. Proc. Soc. Exp.
Biol. Med. 112:1025-1027.

Meizel, S. and S. K. Mukeyi, 1975. Proacrosin from rabbit
epididymal spermatozoa: Partial purification and
initial biochemical characterization. Biol. Reprod.
13:83-93.

Mittal, K. K. 1975. Human histocompatability (HC-A) anti-
gens in semen and their role in reproduction. Fertil.
Steril. 26:700-703.

Pace, M. M. and E. F. Graham. 1974. Components in egg yolk
which protect bovine sperm during freezing. J. Anim.
Sci. 39:1144-1149.

Phillips, P. H. The preservation of bull semen. 1939.
J. Biol. Chem. 130:415-

Polge, G., A. V. Smith and A. S. Parkes. 1949. Revival of
spermatozoa after vitrification and dehydration at low
temperatures. Nature (Lond.) 164:666.

56

Pursel, V. G., L. A. Johnson and G. B. Rampacek. 1972.
Acrosome morphology of boar spermatozoa incubated
before cold shock. J. Anim. Sci. 34:278-283.

Roussel, J. D. and A. R. Austin. 1967. Preservation of

primate spermatozoa by freezing. J. Reprod. Fert.
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Roussel, J. D. and C. R. Austin. 1968. Improved electro-
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Rowe, A. W. 1966. Biochemical aspects of cryoprotective
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Saacke, R. G. and J. M. White. 1972. Semen quality tests
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3) REPRODUCTIVE PERFORMANCE IN MACACA FASCICULARIS

 

FOLLOWING EXTENSIVE LAPAROSCOPY

57

58

REPRODUCTIVE PERFORMANCE IN MACACA FASCICULARIS
FOLLOWING EXTENSIVE LAPAROSCOPY

 

Abstract

Thirty-six mating trials were conducted over an eleven

month period with sixteen adult female M, fascicularis that

 

had been extensively laparoscoped for characterization of
follicular morphology and determination of ovulation. No
difference was observed in menstrual cycle length when
laparoscopy was performed versus cycles during which no
laparoscopic examinations were conducted. Five (31.3%) of
the females in this study conceived. Three gave birth to
healthy live infants. Two abortions from unknown causes
occurred at 112 and 123 days gestation to animals which had
been laparoscoped 43 and 54 times previously. Three females
delivering live infants had been laparoscoped 38, 47 and 49

times prior to breeding.

59

Introduction

 

The present cost of captured primates precludes the
practical use of captive bred animals for research. The
continued depletion of feral populations coupled with
stringent export restrictions by exporting countries in-
creases the necessity for captive breeding programs.
Numerous factors influence the reproductive performance of
captive subhuman primates. Included among these are adapta-
tion to the captive environment, seasonal effects, and the
type of mating strategy.

Hendrickx and Kriewaldt (1969) reported that an eight
month adaptation time was required before menses reappeared
following introduction of baboons (Egpip_§pp) into the
laboratory. Harrison and Dukelow (1973) observed a nine
month adaptation period after introduction to the colony

before squirrel monkeys (Saimiri sciureus) would respond to

 

an ovulatory regime. An increase in conception rate follow-
ing increasing length of residence in captivity has been
reported for three species of macaques (Valerio et al.,
1975).

Seasonal effects on reproduction are extremely pro-
nounced in feral primate groups. Carryover of this effect
to captivity has been observed for several years and can
Significantly contribute to decreased birth rate in captiv-

ity (Niemann, 1970).

60

The length of time that the female is housed with the
male influences the conception rate. Grauwiler and Brugge-
mann (1972) reported a pregnancy rate of 5.1 exposures per

conception using stumptailed macaques (Macaca arctoides) in

 

timed mating trials with male to female exposure up to one

hour. Macdonald (1971) obtained M. arctoides pregnancies

 

after a mean of 1.8 i 0.3 exposures when a five day exposure
time was allowed.

Many nonhuman primate cycles are characterized by the
lack of any outward Sign of estrus. Of extreme importance
in any timed-mating research breeding program is the ability
to accurately predict ovulation. Follicular morphological
development and ovulation has been characterized through
laparoscopic observation in the cynomologus macaque (M,

fascicularis) (Jewett and Dukelow, 1972; Rawson and Dukelow,

 

1973). Data available on the effect of extensive laparo-
scopy on the ability of nonhuman primates to conceive and
deliver healthy infants is limited. Rawson and Dukelow

(1973b), reported the case of a Single M. fascicularis sub—

 

jected to extensive anesthesia, bleeding and laparoscopy
over a 33 day period. Mating and conception occurred on the
17th day and laparoscopy was again performed on days 4 and 7
of pregnancy. A normal infant was delivered at term.

The objective of this study was to evaluate the effect
of extensive laparoscopic examinations on subsequent repro-

ductive performance in M. fascicularis.

 

61

Materials and Methods

 

Sixteen female M. fascicularis maintained at the

 

Endocrine Research Unit (Michigan State University) were
used in this study. They were maintained on a 12 hour
1ight:dark cycle. Temperature was regulated between 21-26°C
with relative humidity fluctuating between 40 and 60% depend-
ing on the season. All animals received a commercially
prepared monkey diet (Wayne, Allied Mills Inc.) and had
unlimited access to water.

The macaques were individually housed in 122 cm stain-
less steel, flush-type cages equipped with slotted floors,
perches and squeeze back devices to facilitate handling.
Menstruation had been recorded daily over the last 8 years.

Seven adult male macaques were screened for motile
sperm concentrations obtained both through electroejaculation
or through vaginal aspiration after mating. Based on this
screening, five males were selected for use in this study.

For laparoscopic examination an intramuscular injection
(10 mg/kg body weight) of Ketamine-HCl (Vetalar, Parke-Davis)
was used to induce anesthesia. The monkeys were placed on
an inclined table with their feet elevated and secured with
adjustable straps (Jewett and Dukelow, 1973). The abdomen
was Shaved and prepared with benzalkonium chloride (Zepher-

ian, Winthrop, New York). A trocar-cannula was inserted

62

through a 8-10 mm incision in the abdomen. Following inser-
tion the trocar was withdrawn and replaced with the laparo-
scopic telescope (135°, 5 mm. pediatric model, Richard Wolf
Co., Knittlingen, W. Germany). A flexible fiber optic

cable connected to a Wolf model 4000 light source was used
to illuminate the abdomen through the laparoscope. A Verres
cannula was inserted 5-6 cm. lateral to the laparoscopic
insertion point as a probe for manipulation of the ovaries.
The abdomen was insufflated with 5% COZ/air.

After completion of the examination, the Verres cannula
and laparoscope were withdrawn leaving the cannula in place
to allow escape of the insufflation gas. The cannula was
then removed. Furacin powder was applied and the skin
incision closed with a single suture (0000 chromic gut,
Ethicon). Penicillin -G(150,000 units) was then adminis-
tered to combat infection.

Laparoscopic observation of follicular development and
the stage of the menstrual cycle dictated the time of intro-
duction of each female into a cage with a male. Mating
trials were conducted from as early as day 2 of the cycle
until just before ovulation as determined by laparoscopic
observation. Mating periods lasted from several hours to

several days.

63

Results

Breeding trials were conducted over an eleven month
period (Table 9). Five (31.3%) of the females conceived in
36 trials. Three females gave birth to healthy live infants.
Two abortions occurred at 112 and 123 days of gestation to
animals which had been laparoscoped 43 and 54 times previ-
ously. Gross anatomical observation of the fetuses did not
reveal the cause of the abortion of these pregnancies.
Three females delivering live infants had been Laparoscoped

38, 47 and 49 times prior to mating.

Discussipn and Conclusion

 

Extensive use of laparoscopic examination of follicular
development maximizes the utility of nonhuman primates for
reproduction research. The technique of laparoscopy offers
a feasible alternative to repeated laparotomy which results
in abdominal adhesions after only 3 or 4 procedures. While
adhesions can occur after extensive laparoscopy (generally
in excess of 30) they are largely avoided by careful tech-
nique. Laparoscopic electrocoagulation and cautery can
correct adhesions that do occur.

Laparoscopy is a reliable means of providing access to
the internal organs of expensive primates such as the

chimpanzee, enhancing the value of this experimental animal

64

 

 

 

Table 9. Summary of Results of Mating Trials Conducted
Between May 1, 1975 and March 18, 1976
Days of
Number of Exposure to
Laparoscopic Mating Male Resulting
Animals Observations Trials in Pregnancy Results
7 43 3 5 Abortion-
(112 days
9 44 -
11 31 -
12 54 6 Abortion-
(123 days)
17 45 2 -
31 32 1 -
39 47 l 6 Livebirth
40 67 l -
41 38 2 6 Livebirth
42 44 2 -
43 47 3 -
44 64 6 -
51 82 2 -
53 49 2 4 Livebirth
54 43 3 -
55 32 l -

 

65

model (Graham, 1976). Bosu (1973) observed no complications
following over 100 laparoscopic examinations during the
normal menstrual cycle of the rhesus monkey. Laparoscopic
‘observation was used in 16 rhesus monkeys during 27 menstrual
cycles (Dierschke and Clark, 1976). Cycle length was 27.5
i 0.6 days in cycles during which laparoscopy was performed
compared to 28.9 i 0.6 days and 28.9 i 0.8 days in cycles
before and cycles after laparoscopy was performed (a non-
Significant difference). No difference was observed in the
cycle length of control animals in which no laparoscopic
examinations were conducted (27.8 i 0.2 days). Laparoscopy
also produced no change in the length of the follicular or
luteal phase in animals undergoing laparoscopy when compared
to control animals. This is in agreement with results re-
ported by Dukelow (1977) in which the laparoscopic examina-

tion of M. fascicularis during 252 cyles resulted in a cycle

 

length of 30.6 i 5.0 (Mean : 8.0.) days was observed as
compared to menstrual cycle length of 29.4 i 11.1 days in
334 cycles in which no laparoscopies were performed.

The results of the present study demonstrate that non-
human primates retain the capacity to conceive and deliver
normal live young after extensive laparoscopic examinations.
It is concluded that extensive laparoscopy does not inter-
fere with the mechanism of ovulation, menstrual cycle length

or normal pregnancy in M. fascicularis.

 

66

References

 

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nation of ovaries in the rhesus monkey. J. Med. Primat.
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Dierschke, D. J. and J. R. Clark. 1976. Laparoscopy in
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Dukelow, W. R. 1975. Morphology of follicular development
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Dukelow, W. R. 1977. Ovulatory cycle characteristics in
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tion and laparoscopic aspiration techniques in Macaca
fascicularis. J. Medical Primatology 2:103-113.

 

Macdonald, G. J. 1971. Reproductive patterns of three
Species of macaques. Fertil. Steril. 22:373-377.

Niemann, W. H. 1970. Comparative aspects of reproduction
in primates. Proc. 3rd Inter. Cong. Primat. (Karger,
Basel) 1:134-237.

67

Rawson, J. M. R. and W. R. Dukelow. 1973a. Observation of
ovulation in Macaca fascicularis. J. Reprod. Fert.
34:187-190.

 

Rawson, J. M. R. and W. R. Dukelow. .1973b. Effect of
laparoscopy and anesthesia on ovulation, conception,
gestation and lactation in a Macaca fascicularis.
Lab. Primate Newsletter 12:4-5.

 

 

SUMMARY AND CONCLUS IONS

Macaca fascicularis was used as a model nonhuman prim-

 

 

ate for investigation of seasonal effects on reproductive
variables in the male. A technique for semen collection was
developed and semen extender components and preservation
techniques were altered to determine their effect on the
motility of cynomologus macaque sperm after pellet freezing.
Breeding trials were conducted to determine the effects of
extensive laparoscopic examination on subsequent reproduc-

tive performance in the female M. fascicularis. The follow-

 

ing conclusions are drawn from the experimental data
obtained in these studies:
1. The effect of season on the annual variation of
reproductive characteristics is markedly reduced

in the laboratory housed male M. fascicularis.

 

2. Semen collection using a rectal probe for electro-
ejaculation is a rapid, reliable method of obtaining
motile Spermatozoa for reproductive studies in the

male cynomologus macaque.

3. A wide range of egg yolk concentration are efficient

as cryoprotectants in semen preservation.

68

 

 

69

Semen extender pH in the range of 7.2 to 8.0 provide

the best post thaw motility.

Cynomologus macaque sperm have a definite require-
ment for glycerol and that levels in the neighbor-
hood of 7-10% are the most effective in maintaining

motility after freezing.

Extremely Short equilibration times of sperm with
glycerol prior to freezing result in maximum sperm

motility after freezing.

Extensive laparoscopy does not interfere with the
mechanism of ovulation, menstrual cycle length or

normal pregnancy in M. fascicularis.

 

PUBLICATIONS BY THE. AUTHOR

Seasonal Reproduction in the Laboratory Housed Male

Cynomologus Macaque, Macaca fascicularis, by James P.

 

Mahone and W. Richard Dukelow. Journal of Reproduc-

tion and Fertility, 1977 (submitted).

Semen Collection and Preservation in Macaca fascicularis,

 

by James P. Mahone and W. Richard Dukelow. Fertility

and Sterility, 1977 (submitted).

Reproductive Performance in Macaca fascicularis Following

 

Extensive Laparoscopy, by James P. Mahone and W.
Richard Dukelow. Journal of Medical Primatology, 1977

(submitted).

70

Name:

Born:

VITA

James Patrick Mahone

October 29, 1945

Birthplace: Staten Island, New York, N. Y.

Formal Education: Glendale High School

Glendale, Arizona

U. 8. Coast Guard Academy
New London, Connecticut

University of Arizona
Tucson, Arizona

Michigan State University
East Lansing, Michigan

Degrees Received: Bachelor of Science

U. S. Coast Guard Academy, 1967

Member of: American Primatological Society.

Honors:

Recipient of the Engineering Design and Analysis
Award for the Outstanding Engineering Project,
1967.

Recipient of the Sigma Xi Award for Outstanding
Graduate Research, 1967.

71

 

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