Hormonal stimulation and embryo collection in the domestic cat (Felis catus )

THERIOGENOLOGY

HORMONAL STIMULATION AND EMBRYO COLLECTION IN THE DOMESTIC CAT(FELIS CATus) -B.L. Dresser,1

C.S. Sehlhorst, l,3 K.B. Wachs, 1 G.L. Keller,2 E.J. Gelwicksl and J.L. Turner1

lcincinnati Wildlife Research Federation (Cincinnati Zoo and Botanical Garden, Kings Island Wild Animal Habitat, University of Cincinnati College of Medicine) Cincinnati, OH 45220 2University of Cincinnati College of Medicine, Department of Laboratory Animal Medicine Cincinnati, OH 45267-057 1 Received for publication

:

Accepted:

December 24, 1986 September 25, 1987

ABSTRACT Sexually mature domestic cats (Felis catus) were treated with one of eight hormone regimens to induce estrus, folliculardevelopment and superovulation. Queens were allowed to breed naturally following hormonal stimulation, and laparotomy was utilized to observe ovarian response and to perform uterine flushes for embryo recovery. The results indicate that it is possible to perform successful repeated surgical collections of transferable quality embryos with the domestic cat. The regimen which evoked the most desirable results in terms of ovarian response and embryo recovery was a subcutaneous injection of follicle stimulating hormone-pituitary (FSH-P) once every 24 h for 6 d in the amount of 0.75 mg/d for the first 5 d and 0.25 mg on the 6 d, followed by two 375 IU intramuscular injections of human chorionic gonadotropin (hCG) 24 h apart, beginning on the same day as the sixth injection of FSH-P. Key words: domestic cat, superovulation, uterine flush

embryo collection,

induced estrus,

Acknowledgments The authors wish to thank Dr. Carmela Skillman for her advice and assistance in the comparative statistical analysis of data and Vickie Stidham for her help in assembling this manuscript. This project was funded in part by the Institute of Museum Services and Nixon Griffis Fund for Zoological Research. of Cincinnati College of Medicine, University 3Present address: Department of Anesthesia, Cincinnati, Ohio 45267-0531.

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INTRODUCTION

Although intra and interspecies embryo transfers have proven successful in there have been no reports of successful exotic bovids, equids and primates, Reed et al. (1) surgically collected two morulae embryo transfer in exotic felids. that appeared viable from one Bengal tiger (Panthera tigris) that had been previously treated with FSH-P and hCG. Subsequent transfer of these embryos to an FSH-P and hCG synchronized African lion (Panthera lea) did not result in offspring. Bowen et al. (2) reported five separate laparotomz in a natural estrus African lion induced to ovulate with hCG or gonadotropin releasing hormone (GnRH). A total of three embryos were collected and frozen for future transfer. It is generally felt that a better understanding of the feline reproductive cycle and its response to artificial manipulation is needed before proceeding further with embryo transfer in exotic felids. Because of the similarity between the reproductive anatomy of the domestic cat and most wild felids (3), this result can best be achieved through studies involving the domestic cat as an animal model. Some information has been reported in the literature regarding the hormonal manipulation of the estrous cycle and the recovery and transfer of embryos in the domestic cat. Kraemer et al. (4) reported successful surgical collection and transfer of preimplantation embryos in naturally cycling domestic cats. An average of 2.8 embryos per cat were collected in 17 collections. All embryos collected were subsequently transferred. Although pregnancies were diagnosed in four of nine recipient cats, only four offspring resulted from 47 embryos transferred. Data on hormonally induced estrous behavior, follicle development and ovulation in the domestic cat were published by Wildt and Seager (5). Examining the use of varying doses of hCG in the invocation of an ovulatory response in the noncopulating naturally cycling domestic cat, they found that the proportion of mature follicles ovulating is an increasing function of graded doses of exogenous hCG. Wildt and Seager (5) concluded that the maximum ovulatory response in their study was achieved through an intramuscular injection of 500 IU hCG on Day 1 of observed estrus or on both Day 1 and Day 2 of estrus. In a study of exogenous gonadotropin induced cyclicity in the domestic cat, Wildt et al. (6) demonstrated that both follicle stimulating hormone (FSH) and pregnant mare’s serum gonadotropin (PMSG) were effective in inducing follicle development and estrous behavior, but the high incidence of premating follicle rupture in PMSG-stimulated cats made FSH the hormone of preference. A comparison of varying doses of single and multiple injection regimens of FSH demonstrated that the multiple injection regimen produced more consistent results in terms of estrous behavior and ovulation rate, but also led to a hypersensitive response which resulted in large follicular cysts on the ovaries which failed to rupture. Wildt et al. (6) concluded that the most effective hormone regimen for inducing follicle growth in the domestic cat consisted of daily intramuscular injections of 2.0 mg FSH until estrus but not for more than five consecutive days.

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A recent study by Goodrowe et al. (7) examined embryo recovery rate and qualirty in cats experiencing a natural estrus versus eats induced into estrus with 2.0 mg injections of FSH-P daily for 5 d. Although the number of embryos collected per cat was slightly greater in induced estrus cats than in natural estrus cats (4.4 f 0.7 vs 3.2 + 0.4, respectively), the induced estrus cats in that study showed a much greater occurrence of unovulated follicles and unfertilized ova as well as a smaller proportion of transferable quality embryos. This study was undertaken to develop a hormonal regimen which would consistently follicular development and result in the onset of estrus, superovulation without ovarian hyperstimulation and cystic follicles, and to develop techniques for the repeated, successful collection of large numbers of transferable quality embryos from domestic cats. It is hoped that the information about ovarian stimulation and embryo collection gained from this study will contribute to the development of similar techniques for exotic felids. MATERIALS AND METHODS Animals Forty-six random source, sexually mature female domestic cats were partially conditioned by the supplier 4 wk prior to receipt with vaccinations against feline panleukopenia, rhinotracheitis and calici virus, as well as treatment Upon receipt and again 2 wk thereafter, the for internal and external parasites. cats received a booster vaccination of Premune RCPN (feline rhinotracheitiscalic:+panleukopenia-pneumonitis vaccine)a, pyrantel pamoate (Strongid-T)b as an Treatment for all anthelmintic, and praziquantel (Droncit)c as a cestocide. Only following a 3 to 4 wk post external parasites was given when indicated. receipt conditioning period were animals initiated into the study. They were housed in colonies of five to eight queens or caged individually for purposes of observation, pre- or postoperative care. Dry cat chow (Purina) and water were A 14-h fluorescent light cycle was maintained year round. provided ad libitum. However, some cats were in a room where two small windows provided limited Room temperature fluctuated between exposure to natural lighting conditions. 60°F and 85oF, depending on seasonal weather conditions. Blood samples were drawn every 3 mo or less for feline leukemia testing and determination of serum total protein. All queens were examined for detection of classic estrous behavior (vocalization, rolling, lateral tail deflection, lowered forelimbs and arched hindquarters and treading) several times a week. Queens suspected to be in heat were usually placed with a tom to check the tom’s interest in mounting and the queen’s receptivity; however, intromission by the male was never allowed to occur. Continuous estrous behavior records were kept for each queen. Queens were selected for each procedure according to their overall health, the time elapsed since their last use in the study and the time elapsed since aBurroughs Wellcome Co., Kansas City, MO. bpftizer, Inc., New York, NY. CBayvet Division, Miles Laboratories, Shawnee, KS.

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behavioral estrus was observed. As a general rule, hormone injections were never begun with any queen who was thought to have been in heat during the previous 7 to 10 d. Queens in this study were used from one to five times. Of the queens which were used more than once, the time between procedures averaged 15.3 wk. Queens

were fasted

for at least

15 h prior to surgical

procedures.

Superovulation Listed (Table 1) are the eight different hormone regimens tested in this In each case, estrus was induced by subcutaneous injections of FSH-Pd study. were equal and the sixth given every 24 h for 6 d. The first five injections injection was one-half to one-third the dosage of the initial five injections. FSHP was diluted to concentrations of 0.5, 1.0 or 2.0 mg/ml with normal saline. Newly reconstituted FSH-P was aliquoted and stored frozen at -1OOC. Frozen FSH-P was thawed slowly prior to use and kept at 3 to 5OC for no longer than 5 d postthaw; the unused portion was discarded. Except for Regimen I, ovulation was induced by two intramuscular injections of hCG (10,000 U.S.P units/l0 ml vial)e 24 h apart, beginning on the same day as the sixth injection of FSH-P. Reconstituted hCG was stored at 3 to 5OC until discarded after 30 d. Following initial administration of hCG, queens were placed in a tomcat’s cage and usually remained there until the day prior to embryo collection to allow Observed breedings were logged. Actual maximum opportunity for copulation. daily observation time was approximately 2 h. Surgery Surgical embryo recovery was performed 11 to 13 d after the first day of FSH-P administration. For purposes of surgical embryo collection, anesthesia was induced with ketamine hydrochloride (Ketaset)f intramuscularly at 10 to 12 mg/lb body weight, atropine sulfate intramuscularly at 0.2 to 0.3 ml (l/120 g/cc) and topical administration of lidocaine hydrochloride 1% (w/v) in the pharyngeal area Methoxyflurane (Metafane)g and oxygen to facilitate endotracheal intubations. were used for maintenance of anesthesia. Following ventral midline incision, ovaries were exteriorized for evaluation and enumeration of unovulated follicles and ovulation sites. The uterine body was occluded anterior to the internal cervical OS with a Cooley type clamp,h then The uterine horns catheterized with a media primed, 14-gauge i.v. catheter.’ The horn opposite the one to be were flushed separately in the following manner: flushed was occluded at its posterior pole with a Cooley type clamp.

dBurns-Biotec Laboratories, Inc., Omaha, NB. eLypho-Med, Inc., Chicago, IL or Carter-Glogau fBristol Laboratories, Syracuse, NY. gpitman-Moore, Washington Crossing, NJ. hLawton Surgical Instruments, Madison, CT. iCritikon, Tampa, FL.

Laboratories,

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Inc., Glendale,

AZ.

1987 VOL. 28 NO. 6

13 7 10 22

13

13

13

11

10

22

2

3

4

5

6

7

11 to 13 11 to 13 12 to 13

Days 6 to 7 = 750 III/d Days 6 to 7 = 375 III/d Days 6 to 7 = 750 W/d Day 6 = 500 IU Day 7 = 750 IU Days 6 to 7 = 750 III/d

Days 1 to 5 = 0.5 mg/d Day 6 = 0.25 mg Days 1 to 5 = 0.75 mg/d Day 6 = 0.25 mg Days 1 to 5 = 0.75 mg/d Day 6 = 0.25 mg Days 1 to 5 = 1.0 mg/d Day 6 = 0.5 mg Days 1 to 5 = 1.0 mg/d Day 6 = 0.5 mg

8

10

9

---._--__

13

12

12 to 13

12

Days 6 to 7 = 350 W/d

Days 1 to 5 = 0.5 mg/d Day 6 = 0.25 mg

12

Ova recovery daya

None

hCG dose

Days 1 to 5 = 0.5 mg/d Day 6 = 0.25 mg

FSH-P dose

Day 6 = 500 IU Days 1 to 5 = 1.5 mg/d Day 7 = 750 IU Day 6 = 0.5 mg _-_-_---II--_--I__ ------I_---_-_---aNumber of days following first injection of FSH-P.

13

12

10

13

1

No. of queens flushed

No. of queens treated

Regimen no.

Table 1. Hormone regimens employed in this study

l-HERIOGENOLOGY

A five-inch length of plastic tubing (Venopak SO)j was primed with media and connected to the distal point of the catheter, while the opposite end was placed in a 90 x 50 mm Pyrex scanning dish for collection of fluids. Media was slowly introduced via a blunted 18-gauge needle and 12 cc glass syringe at the anterior tip of the horn, then retrieved in the scanning dishes (Figure 1). Approximately 100 ml of media warmed to 37OC were infused through each horn. Uterine flushing media was supplemented Dulbecco’s phosphate buffered Supplement consisted of saline (D-PBS) with 2% newborn calf serumk added. glucose (1.0 g/l), sodium pyruvate (0.4 g/l), glutamine (0.146 g/l), penicillin-G (0.063 g/l), streptomycin (0.05 g/l) and sodium bicarbonate (0.098 g/l). All reagents used for supplement were for tissue cell culture. Any ova found in flushing media were transferred within 30 min after collection via Monoject tomcat catheter1 into D-PBS (modified with the same supplement described for the flushing media) with 15% newborn calf serum added. Ova were classified according to stage of development and evaluated with both stereo and inverted microscopes. Embryos were subjectively graded as excellent, good, fair, poor and dead or degenerate based on the integrity of the zona pellucida and inner cell mass, and their development in culture. Embryos were considered to be of transferable quality as long as they were not graded as dead or degenerating. Catheterization to the reproductive

and needle tract.

puncture

sites

were not sutured

to lessen

trauma

Heparinized Ringer’s solution or saline was used for frequent drenching of the surgical site. Heparinized dexamethasone saline was used for extensive lavage of the abdomen in donor queens before closure to ensure maximum dilution of any remaining blood in an effort to deter formation of adhesions. Both drenching and lavage fluids were warmed in a 450 C waterbath prior to use. All glassware and needles used in media preparation and embryo collection were put through a vigorous embryo wash in 7X Linbro soapm using deionized feed water that was further filtered through a mixed bed deionizer and organic removal filter and finally filtered through a Pyrogard (trademark pending) ultrafilter.” Following embryo wash, all equipment was thoroughly rinsed, air dried, packed and exclusively autoclaved. Analysis

of Data

Data on ova recovery and ovarian response were statistically analyzed using one-way analysis of variance (ANOVA) for normally distributed variables (parametric) or Kruskal-Wallis for variables not distributed normally (nonparametric). A value of p 2 0.05 was considered statistically significant. A Newman-Keuls range test was applied to determine statistical difference between means. -jAbbott Laboratories, Chicago, IL. kGibco Laboratories, Grand Island, NY. ISherwood Medical, St. Louis, MO. mFlow Labs Inc., McLean, VA. “Millipore Laboratory Products, Bedford, MA.

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Figure

1. Illustration

of surgical

embryo

flush technique

utilized

in this study.

RESULTS Presented (Table 1) is a summary of the FSH-P and hCG doses employed in the eight hormone regimens examined in this study, as well as the number of queens treated and flushed in each regimen and the day or range of days of ova recovery utilized within each regimen. Ovarian response and ova recovery results from the eight hormone regimens are likewise reported (Table 2). This study consisted of 105 laparotomies. Nine of 105 did not include a uterine flush, seven of these because of little or no observed ovarian response and two because of the observation of purulent material within the reproductive tract. From 96 flushes, 1264 ova, including both fertilized ova and unfertilized ova (UFO), were colIected, producing an average of 13.2 ova per queen flushed. Although not statistically significant (p = 0.13), the highest number of ova collected was with Regimen 4 (23.7 2 4.9) and the lowest number of ova collected was with Regimen 1 (6.4 f 3.1). The most ova collected from any one queen was 75 (Regimen 3) and the least collected was 0. No ova were collected on 19 occasions, and these 19 occasions were distributed among all the regimens except

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THERIOGENOLOGY

Regimens 4 and 5. The unsuccessful collection of ova in 19 cases cannot be The average number of ovulation sites and attributed to a poor ovarian response. follicles noted per queen in the 19 cases was 14.4 and 9.6, respectively. Over onehalf of the 19 unsuccessful cases appeared to be attributable to a flush which could not be properly executed due to damage to the tract from previous surgeries It is believed that late breedings or problems with the placement of the catheter. which may have left the ova in the oviducts at the time of laparotomy was another contributing factor in some of these cases. It was determined that Regimen 5 resulted in the collection of a statistically significantly greater number of UFO than Regimens 1, 6 and 8. The greatest number of UFO collected per queen was 12, and this occurred twice. No UFO were collected in 69% of the queens flushed. There was no statistically significant difference among the eight regimens for number of fertilized ova (embryos) recovered and number of transferable quality embryos recovered. However, Regimen 4 had the largest number in both categories (23.0 + 4.9 and 15.8 + 4.2, respectively) and Regimen 1 had the smallest number in both categories (6.4 + 3.1 and 5.9 f 2.8, respectively). The largest number of transferable quality embryos collected from any one queen was 48 (Regimen 3). No transferable quality embryos were collected in 35% of all queens flushed. The 34 occasions when no transferable quality embryos were collected were fairly evenly distributed among all eight regimens. The number of ovulation sites counted on both ovaries for queens treated under Regimen 4 was 30.9 f 3.5, a value that was significantly larger than the number of ovulation sites noted in Regimens 1 and 5. The average number of ovulation sites counted per queen was 19.6. Only five queens out of 105 exhibited a total lack of ovulation sites upon laparotomy, and 91% of all queens examined exhibited more than three ovulation sites. An individual ova recovery rate was computed for each of the 96 flushes by dividing the number of ova collected by the number of ovulation sites counted. A group ova recovery rate was then computed for each of the eight regimens by averaging the individual ova recovery rates within each regimen. The group ova recovery rates ranged between 81.5 + 16.8% in Regimen 4 to 28.9 + 11.1% in Regimen 8, but the differences among the regimens were not statistically significantly different from one another. Queens treated under Regimen 1 exhibited a unovulated follicles on both ovaries than Regimens of follicles counted per queen was 9.7. The largest any queen was 53 (Regimen 8). Fourteen percent observable follicles at the time of laparotomy.

significantly smaller number of 6 and 8. The average number number of follicles counted on of all cats in the study had no

Of the 96 uterine flushes in this study, six occurred on the eleventh day following the first injection of FSH-P, 75 occurred on the twelfth day following first injection, and 15 occurred on the thirteenth day following the first injection. Although there were no statistically significant differences among the numbers of ova collected on the three different days of ova recovery, there was a trend toward greater numbers of ova collected with a later recovery day (Table 3).

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13.9 + 5.8

14.5 IL 4.4

9.5 + 2.1

9.1 ? 3.9

5

6

7

8

a_+SEM.

0.13

0.4 -+ 0.3

23.7 + 4.9

4

P-value

1.2 + 0.4

15.7 -I 6.0

3

0.03

0.2 f 0.1

3.2 + 1.8

0.7 f 0.3

0.8 + 0.5

2.5 + 1.0

12.9 f 3.7

2

0

6.4 + 3.1a

Total ova

Unfertilized ova (UFO)

and ovarian

as a function

10.9 + 3.1 10.2 + 3.1 7.6+

15.6 k 2.8

17.8

63.42

81.5 + 16.8 80.5 f 39.3

30.9 f 3.5

0.11

0.11

0.02

28.9 f 11.1 20.5 + 4.0

7.9 * 3.5

8.7 _+3.6

0.31

16.9 * 4.8

11.5

59.2+17.6 + 1.6

4.3 i: 1.2

8.3 + 2.1

0.01

6.6 + 2.0

10.6

55.4?

21.8 f 4.7

11.3 + 4.4

14.3 f 4.4

2.1

16.2 +- 4.6

5.3 +- 2.9

10.7 +_5.5

3.0

13.1 + 3.8

61.5 + 12.1

18.6 + 3.6 21.02

2.7 + 1.3

Unovulated follicles

36.1 + 14.3

Ova recovery rate (%)

regimens

11.8 ? 3.4

Ovulation sites

of hormone

15.8 f 4.2

8.9 ?I 3.9

5.8 f. 1.9

5.9 f 2.8

Transferable quality embryos

response

23.0 + 4.9

14.9 +_6.1

10.4 f 3.4

6.4 f 3.1

Fertilized ova (embryos)

Group mean + SEM for ova recovery

1

Regimen

Table 2.

2

rl

THERIOGENOLOGY

The breakdown of usage for the 46 queens that were hormonally treated as donors in this study was as follows: 12 queens were used once; 15 were used twice; 14 were used three times; four were used four times; and one was used five times. Four of the 46 subjects had been hormonally stimulated as donors in a pilot study at least once prior to the time they were initiated into this study. In addition, nine of the 46 subjects had been hormonally treated to serve as recipients for embryo transfer at least once prior to the time they were initiated into this study. Presented (Table 4) are the ova recovery and ovarian response data as a function of the number of times an animal had been cycled as a donor and received hormonal stimulation for that purpose. The table presents averages for all first, second, third, fourth and fifth time donors. Few differences can be detected in the ova recovery and ovarian response data among the queens which were Set up as donors for the first, second and third time. A sharp decrease in ova recovery occurred in queens undergoing their fourth donor laparotomy; however, the ovarian response data remains fairly consistent. This suggests that the decrease in ova recovery might be attributable more to trauma within the reproductive tract from past surgeries rather than a refractory response to the hormones. A sharp reduction in both ovarian response and ova recovery occurs in the figures for fifth time use; however, this data should not be considered conclusive because it is derived from only one queen who had been a fair to poor performer in each of her previous four usages. There was a slight trend toward a larger number of ova collected better ova recovery rate in queens who had been rested for at least between surgeries, but this difference was not statistically significant. A similar of ova collected

and a 12 wk

statistically nonsignificant trend toward slightly greater numbers was also noted as the weight of the donor queen increased.

DISCUSSION A wide range of occlusion and catheterization techniques were evaluated to determine the procedure that permitted the most effective collection of embryos with the least amount of trauma to the reproductive tract. Early attempts at surgical embryo recovery by these authors frequently resulted in the leakage of flushed fluids around the catheter site and the possible loss of embryos in these The technique and instruments fluids as well as unnecessary trauma to the tissue. listed in the Materials and Methods section of this paper are those that have proven most effective after many trials. It must be emphasized that if repeated use of the same queen is anticipated, then attention should be paid throughout the procedure to restricted and delicate handling of the tissues, especially the ovaries themselves. A sharp decline in adhesions was noticed as the degree of tissue handling decreased. Likewise, a decrease in trauma and adhesions to the urinary bladder was noticed after manual expression of the bladder prior to surgery was discontinued. The tone of a queen’s uterine horns proved to be a fairly reliable indicator of responsiveness to the hormone treatment. If the horns showed good tone at laparotomy (firm, thickened walls and a ridged, tortuous appearance), the ovarian

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0 ;

0

0

a:

0

0

0

0

5

2 r+

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response was usually quite good. Poor tone, evidenced by a smooth, flaccid feel to the uterine horns, usually was seen in queens that did not respond as well to the hormone treatment. When reporting the number of unovulated follicles present on the ovaries, they were ranked according to size. To minimize handling of the ovaries, the size ranking was subjective rather than by actual measurement. A rough standardization of the sizes would be as follows: 2 mm or less = small; 3 to 5 mm = medium; and over 5 mm = large. Occasionally small (< 2 mm) follicular structures not protruding above the ovarian surface were observed. These were not included in the total follicle count. The relative sizes of all follicles observed in this study were ranked as follows: 52% small, 36% medium and 12% large. One curious occurence in our study was the total absence of ovarian response at laparotomy in three queens that were simultaneously treated under Regimen 3. Because each of these queens had responded to hormone treatment either prior or subsequent to that time, it was felt that this total lack of stimulation had to have been caused, at least in part, by human error. Other than these three queens, there were only four occasions when the ovarian response was deemed to be too poor to proceed with a flush. If embryos were recovered, they were generally, the first 25 ml of flushing media which was collected.

but not always,

found

in

Throughout the study, embryos recovered at laparotomy were either transferred immediately into recipients, frozen for future transfer, used for micromanipulation or discarded. Toward the end of the study, some of the recovered embryos were used for culture studies to observe the effects of different media on development. It was noted at this time that a number of embryos which appeared to have the classic signs of death or degeneration at the time of recovery (darkened, disorganized cell masses, an abundance of extruded cells, thick zona pellucidae, presence of lipid droplets) developed in culture. This result is mentioned only because this finding led the authors to believe that embryos previously classified as dead or degenerate during the course of this study may have been misclassified. Prior to this study, a 2.0 mg FSH-P dose was tried on a number of queens in a pilot study. In several instances cystic ovaries were observed similar to those described by Wildt et al. (6), so it was decided to test some lowered doses of FSHP. In the eight hormone regimens included here, four different FSB-P doses (0.5 mg, 0.75 mg, 1.0 mg and 1.5 mg) combined with varying doses of hCG were tested. It was decided to use hCG as an additional stimulus for ovulation in all but one regimen. Regimen 1 did not utilize hCG and, as seen (Table 2), it produced the poorest ovarian response and the fewest number of collectable embryos. In comparing the four FSH-P doses tested, many of the variables examined in our study followed a standard curve, in that the poorer responses came at the low and the high ends (0.5 and 1.5 mg PSH-P) and the more desirable responses ca.me at the middle doses of 0.75 and 1.0 mg FSH-P. Lofstedt (8) has noted that the subjective interpretation of estrus signs demonstrated by queens does not always coincide with hormone profiles or vaginal cytology. We relied on observable signs of estrous behavior. Animals that were

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not observed in heat for the previous 7 to 10 d were chosen to begin hormone treatment. One of the biggest variables in this study was clearly the uncertainty as to the stage of the estrous cycle of the queen when hormone treatment began. To conclude, we found that it is possible to perform successful repeated surgical collections of transferable quality embryos with the domestic cat. All eight hormone regimens tested in this study were found to be capable of stimulating follicular development, ovulation and the production of transferable quality embryos. Although no one hormone regimen stood apart as more uniformly effective than any other when statistically tested, the best results in terms of ovarian response and embryo recovery came from the regimen of a subcutaneous injection of FSH-P once every 24 h for 6 d in the amount of 0.75 mg/d for the first 5 d and 0.25 mg on the 6 d, followed by two, 375-IU intramuscular injections of hCG 24 h apart, beginning on the same day as the sixth injecti.on of FSH-P. REFERENCES 1.

Reed, G., ‘Serringer, ((Panthera ((Panthera

2.

Bowen, M.J., Platz, C.C., Jr., Brown, C.D. and Kraemer, D.C. Successful artificial insemination and embryo collection in the African lion (Panthera .-Leo). Proc. Amer. Assn. Zoo Vets., New Orleans, pp. 57-59 (1982).

3.

Wildt, D.E., Platz, C.C., Seager, S.W.J. and Bush, M. Use of gonadotropic hormones to induce ovarian activity in domestic and wild felids. Proc. Amer. Assn. Zoo Vets., Washington, pp. 44-46 (1980).

4.

Kraemer, D.C., Kinney, G.M. and Schriver, M.D. Embryo transfer domestic canine and feline. Arch Androl. 5:lll abstr. (1980).

5.

Wildt, D.E. and Seager, S.W.J. Ovarian response in the estrual cat receiving varying dosages of HCG. Hormone Res. 9:144-150 (1978).

6.

Wildt, D.E., Kinney, reproductive cyclicity (1978).

7.

Goodrowe, K.L., Howard J.G. and Wildt, D.E. Embryo recovery and quality in the domestic cat: Natural versus induced estrus. Theriogenology -25:156 (1986).

8.

Lofstedt, R.M. The estrous cycle of the domestic cat. & The Compendium on Continuing Education, Univ. of Penn. School of Vet. Medicine, Vol. 4, No. 1, pp. 52-61 (1982).

DECEMBER

Dresser, B., Reece, B., Kramer, L., Russell, P., Pindell, K. and P. Superovulation and artificial insemination of Bengal tigers ti ris) and an interspecies embryo transfer to the African lion -+- . Proc. Amer. Assn. Zoo Vets., Seattle, pp. 136-138 (1981). -leo

in the

G.M. and Seager, W.J. Gonadotropin induced in the domestic cat. Lab. Anim. Sci. -28:301-307

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