Equine Nutrition and Physiology Society REFEREED PAPERS FROM THE 13TH SYMPOSIUM

EVALUATION OF THREE TECHNIQUES (FOLLICULAR ASPIRATION, FOLLICULAR

ASPIRATION AND FLUSHING AND SLICING OF THE OVARIES) FOR RECOVERY OF EQUINE OOCYTES

FROM EXCISED OVARIES

J.C. Vazquez, MVZ, MSc; J.F. Moreno, MSc; R. Hanneman, MSc; J.W. Evans, PhD;

D.C. Kraemer, DVM, PhD.

oocyte recovery rate when using a continuous irrigation aspiration system (60.00%) than when using aspiration with a syringe and needle (14.00%).

The present study was an attempt to develop an efficient technique for oocyte recovery from abattoir derived ovaries. Three different procedures were tested: a) aspiration using a vacuum pump, b) aspiration plus flushing the follicles (A-F) and c) rupture of the follicles by slicing the ovaries in a grid pattern.

MATERIALS AND METHODS

SUMMARY

The objective of this study was to compare three different methods of oocyte recovery from equine ovaries obtained from an abattoir. The techniques used were a) aspiration of follicles, b) aspiration plus flushing of the foUicles and c) slicing of the ovaries in a grid pattern. The number of oocytes recovered from each of the three methods was different (P<0.05). From 235 ovaries processed by the aspiration technique, a mean of .94 __..5 oocytes was recovered per ovary. From 711 ovaries processed by the aspiration- flushing system a mean of 1.69 -+ .46 oocytes per ovary was recovered and from 89 ovaries processed by the slicing technique a mean of 3.55-+.45 oocytes perovary was collected. The effect of the system on the mean time spent recovering each oocyte was not significant. The type of system utilized had a significant effect (P<0.05) on the quality of oocytes. The mean number of grade 4 oocytes (completely or partially denuded) recovered with the slic- ing technique was greater than the mean number of grade 4 oocytes recovered by the aspiration-flushing technique.

INTRODUCTION

Abattoir derived ovaries are a convenient source of oocytes used for in vitro fertilization (IVF) research in the equine as well as in other species. In the equine, the two techniques which have been most often used are aspiration of follicles 1'2 and follicle dissection. 1,a

Aspiration is normally performed with a syringe and 21 ga needle. This procedure can be tedious and time consuming when many ovaries are being processed. Follicle dissection is a more time-consuming procedure, even though the oocyte recovery rate is higher. Ok61ski et al. 1 compared those two techniques and reported 4.00 and 1.54 oocytes per ovary and 93.30% and 33.90% oocytes per follicle when using follicle rupture ar.d follicle aspiration, respectively. Vogelsang et al. 4 ~vorking with mares, reported two different follicle aspiration techniques in situ and found a higher

Authors' address: The Department of Animal Science, College of Agriculture and LifeSciences, Texas A&M University, College Station, Tx. 77843-2471. Acknowledgment: Manuscript TA 31124, Texas Agricultural Experiment SlatJon.

The ovaries used in this experiment were obtained from an abattoir about 2 hours driving distance from the laboratory. The ovaries were collected approximately 30 min after slaughter, placed in zip-lock bags with physiological saline solution and maintained at 32 to 39°C, until arrival at the laboratory. Ovaries were rinsed with fresh sterile physiological saline solution (32°C) before start- ing oocyte collection.

Oocyte Recovery Aspiration System. The aspiration system (Fig. IA) consisted

of a vacuum pump (set at 180 mm Hg), connected through tubing a to a 50 ml sterile conical tube b into which the follicular fluid was collected. The rubber stopper in the top of the conical tube had three holes, one through which was passed a ground joint outer luer tube e to which a 14 ga needle was attached and which was used for aspiration. The second hole was adapted to hold the vacuum line tubing and the third hole was an air entrance which helped to control vacuum pressure. The follicular size was not determined and every aspirable follicle was aspirated. When the conical tube was full, the follicular fluid was filtered using a 75 ktm Em-Con filter, d At least 10 ml of follicular fluid were kept in the filter at all times. After several follicles were aspirated 10 to 15 ml of holding medium (low bicarbonate-TALP medium as described by Parrish et al. 5) were added to the follicular fluid in the filter to dilute it and to diminish the probability of clotting.

The oocytes were held in the diluted follicular fluid and in the filter, at room temperature until all ovaries had been aspirated, or until some evidence of clotting of the follicular fluid was observed. Fluid containing the oocytes was then poured into a 100 x 15 mm sterile round disposable petri dish. e The filter was washed with holding medium at room temperature and the petri dish was searched for the oocytes with the aid ofa stereomicroscope. Oocytes were transferred with a 5 Ixl Drummond pipetter f into a petri dish of holding medium. The oocytes remained there until the search was finished. The oocytes were then washed and classified as described later. aTygon, Fisher, Houston, TX. bFisher, Houston, TX. CLab Products Inc,, Houston, TX. dlmmuno Systems Inc, Em-Tex, Grand Prairie, TX eVWR, Sugar Land, TX. fDrummond. Broomall, PA.

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6

1

Figure 1. Follicular aspiration and flushing apparatus.

Combined Aspiration and Flushing systems. A flushing system was used in conjunction with the pleviously described aspiration system to flush the follicles with holding medium while it was being aspirated (A-F system). The flushing system was designed so that the fluid flowed by gravity through a valve and into the follicle through a 16 or 18 ga needle (Fig. 1 B) Both aspiration and flushing needles were placed simultaneously into the follicle.

Slicing system. After rinsing, each ovary was placed in a 100 mm disposable round petri dish and sliced with a surgical blade in a gridded pattern with the intention of breaking as many follicles as possible. The follicular fluid was poured into an Em-Con filter and treated in the same way as described above for the aspiration system. The sliced ovaries were rinsed in 300 ml of holding medium. After having processed all the ovaries, the medium used to rinse the ovaries was also filtered though an Em-Con filter. Searching was performed in the same way as in the aspiration system.

Oocyte Classif ication Oocytes were washed three times in warm holding medium

and classified into one of four groups according to the quality of the cumulus cells surrounding the oocytes; a) oocytes fully surrounded by compact cumulus cells were considered grade 1 (GI), b) oocytes partially surrounded by cumulus cells and that had a non-disrupted corona radiata were classified as grade 2 (G2), c) oocytes with expanded and/or picnotic cumulus cells were classified as grade 3 (G3) and d) oocytes completely or partially denuded were consid- ered grade 4 (G4).

Table 1. Number of oocytes recovered per ovary using different oocyte recovery periods.

No of ovaries No. of oocytes Mean of oocytes per ovary + SD Range of oocytes per ovary

Aspiration 235 222

.94 = 5 c

A-F 711 1199

1.69 = .46 b

Slicinq 89

332

3.55 = .45 a

.29 -1.46 .96- 2.74 3.14- 4.04

A-F, Aspiration and flushing. Numbers showing different superscripts a r e different (P< 0.001).

Total 1035 1753

1.70 ±.87

.29-4.04

Statistical Analysis Statistical analysis was performed using the SAS System version

6.07,ProcGLM usinga one-way classification model and Tukey'stest. For the independent variable "technique" and the dependent variables: "oocytes per ovary", "time required to process each ovary", "time required to recover each oocyte" and"oocyte grade", the model was Yil = Ix + Ti +Eij where Y = response and T = technique.

RESULTS

Oocyte Recovery Oocyte Recovery Rates Related to the Oocyte Recovery

Technique. There was a difference (p<0.05) in the oocyte recovery rates among the three techniques. The highest recovery rate was obtained by the slicing system which yielded an average of 3.61 oocytes per ovary and a range of 3.14 to 4.04 oocytes per ovary between replications. The aspiration flushing (A-F) system yielded the second highest recovery rate with 1.68 oocytes per ovary and a range of .96 and 2.74 oocytes per ovary between replications. The lowest recovery rate was obtained by the aspiration system which yielded a mean of.94 oocytes per ovary with a range of.27 to 1.46 oocytes per ovary between replications (Table 1). From the 711 ovaries processed with the A-F system, 521 were considered for evaluation of the number of oocytes recovered per follicle. Nine hundred and thirty eight oocytes were found from ] 668 follicles aspirated (57.00% recovery per follicle).

Time Spent Per Oocyte Recovered. The mean time neces- sary to process each ovary was different (P< .05) among the three systems. The mean processing time per ovary was 1.51 minutes for the aspiration technique, 3 minutes for the A-F technique and 5.45

Table 2. Average time required to process each ovary and to recover each oocyte using different oocyte recovery techniques.

Min/ovary ± SD Min/oocyte = SD

Aspiration 1.51=0.31 a 1.9=1.00

A-F Slicinq 3.0=0.6 b 5.45=1.03 c 1.6=0.41 1,49=0,49

A-F, Aspiration and flushing. Numbers showing different superscripts are different (P< 0.05).

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Equine Nutrition and Physiology Society REFEREED PAPERS FROM THE 13TH SYMPOSIUM

Table 3. Quality of oocytes recovered using different techniques evaluated by cumulus cell complexes integrity.

Aspiration Aspiration- Slicing Flushinq

No of ovaries No. of Oocytes Grade 1 Grade 2 Grade 3 Grades 2 + 3 Grade 4

78 95

36.51%

62.46 % 1.02 %be

661 1151

33.32 % 58.09 % 7,72 %

65.81% 0.79 %ac

89 332

30.15% 59.54 %

4.55 % 64.09 %

5.75 %b

Numbers showing different superscripts are different (P<O.05).

minutes for the slicing technique. However, the time necessary to recover each oocyte was not different (P> 0.05) among the three different techniques (Table 2 ) .

Oocyte Classification Related to the Recovery Technique For the aspiration system, oocytes graded as G2 and G3 were not separated, so a new grade consisting in adding G2 plus G3 (G2+G3) was formed for all three oocyte collection systems.

From 95 graded oocytes recovered with the aspiration system, 36.51% were G1, 62.46% were G2+G3, and 1.02% were G4. From the 1151 graded oocytes recovered with the A- F technique, 33.32% were G1, 58.09% were G2, 7.72% were G3, 65.81% were G2+G3 and 0.79% were G4. From the 332 graded oocytes recovered with the slicing technique, 30.15% were G1, 59.54% were G2, 4.55% were G3, 64.09% were G2+G3 and 5.75% were G4. The proportion of G4 oocytes recov- ered using the slicing technique was larger (P< 0.05) than the proportion of G4 oocytes recovered using the A-F technique. No other differences (P> 0.05) were detected (Table 3).

DISCUSSION

The results of the present work indicate that the oocyte recovery rate per ovary obtained with the slicing technique is superior to the AF and aspiration systems, and that the A-F System is superior to the aspiration technique, yielding an average of 3.61, 1.68 and .94 oocytes per ovary, respectively. However, even though the number of oocytes obtained per ovary was higher when using the slicing technique, the time spent processing the ovaries with this technique was longer (P<.05) than the time spent by either of the other two techniques for processing the same number of ovaries. Also the A-F system required more time to process the same number of ovaries than the aspiration technique. Because the results of oocyte recovery rate per ovary and the ovary processing rate per minute suggested differences in efficiency for recovery of oocytes, the average-number of minutes required to recover each oocyte was

analyzed for the three techniques. No differences (P< .05) were found among treatments regarding the efficiency of the oocyte collection. The biological significance of the technique on the number of minutes required to recover each oocyte was not tested, but from a practical perspective the results suggest the use of the aspiration and A-F systems for oocyte recovery if the number of ovaries available is large, and the use of the slicing technique if the number of ovaries available is limited. This recommendation is supported by the following considerations: Slicing the ovaries is more time consuming; presents more opportunities for contamina- tion of the oocytes; and yields lower (P<.05) cumulus cell quality.

Ok61ski et al. 1 reported a recovery rate of 4 oocytes per ovary and a recovery of 93.00% oocytes per follicle when using a technique based on dissection and rupture of the follicle. They also reported a recovery rate of 1.5 oocytes per ovary and 34.00% oocytes per follicle when aspirating follicles using a syringe and needle. Zhang et al. 3 reported that most ovaries would yield 5 to 10 oocytes using the dissection system, while some ovaries would produce up to 20 oocytes. The syringe and needle technique used by Okrlski et al. 1 and the A-F system used in the present study yielded similar recovery rates of oocytes per ovary, However, the mean number of follicles aspirated per ovary was different (4.5 vs 3.2). The dissection technique used by Ok61ski et al. 1 and the slicing technique used in the present study yielded similar results. None of the techniques tested in the present study were as efficient as the dissection technique reported by Zhang et al. a

Palmer et al. 6 aspirated pre-ovulatory equine follicles from live mares using a syringe and needle. They reported a 62.70% oocyte recovery rate per follicle. Vogelsang et al., 4 using an A-F system in live mares, reported a 60.00% oocyte recovery rate from pre-ovulatory follicles. A 25% oocyte recovery rate was reported by Bruck el al., 7 during aspiration and flushing of preovulatory folli- cles of estrous mares using a transvaginal ultrasound-guided tech- nique and a needle and syringe. Cook et al. a reported a mean of 57% recovery rate when aspirating and flushing preovulatory follicles and 18.5% recovery rate when aspirating and flushing diestrous follicles with a transcervical ultrasound-guided technique equipped with a vacuum system. Ok61ski et al., 1 using two different systems in excised ovaries reported 33.90% and 93.30% recovery for aspiration with a syringe and needle and dissection systems, re- spectively. In the present study, the A-F oocyte recovery rate per follicle was 57.00%. The recovery rates per follicle reported in the present work were similar to those reported by Palmer et al. 6 and Vogelsang et al.4 The recovery rates per follicle obtained using the A-F technique were superior to the results reported by Ok61ski et al. 1 (57.00% vs 34.00% respectively) when they used a syringe and needle. These data suggestthat the A-F system is more efficient than the aspiration of follicles using a syringe and needle. On the other hand, the number of oocytes per follicle reported by Ok61ski et at.,1 using the dissection method, is superior to the oocyte recovery rates per follicle obtained in the present study using the A-F system (93.00% vs 57.00% respectively).

Ok61ski et al. 1 reported that 89.00% of the oocytes recovered

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Equine Nutrition and Physiology Society REFEREED PAPERS FROM THE 13TH SYMPOSIUM

by dissection, and 29.30% of the oocytes recovered by a syringe and needle had intact cumulus cell complexes surrounding the oocytes. In the present work, the percentages ofoocytes recovered with intact cumulus cell complexes (30.00% for slicing, 33.30% for A-F, 36.51% for aspiration) were comparable to those reported by Ok61ski et al. 1 when they used the syringe and needle system. However, the percentages of intact cumulus cell complexes ob- tained in the present study, using any of the techniques, were lower than those reported by Ok61ski et al., 1 when they used follicular dissection.

Another important consideration which was, however, not tested in this study is that of oocyte contamination by microogan- isms. Both techniques using aspiration are semiclosed systems. Therefore it seems much easier to avoid contamination of the oocytes using these techniques than when using the slicing tech- nique.

REFERENCES

1. Ok61ski A, Babuski P, Tishner M, et al. Evaluation of mare collection methods and stallion sperm penetration of zona free hamster ova. J. Fleprod Fert., Suppl. 1987;35:191.

2. Hinrichs K. The relationship of follicle atresia to follicle size, oocyte recovery rate on aspiration, and oocyte morphology in the mare. Theriogenology 1991 ;36:157.

3. Zhang JJ, Boyle MS, Allen WR, et al. Recent studies on in- vivo fertilization of in-vitro matured horse oocytes. Equine Vet'. J., Equine Embryo Transfer. Suppl. 1989;8:101.

4. Vogelsang MM, Kreider JL, Bowen MJ, et al. Methods for collecting follicular oocytes from mares. Theriogenology 1988;29:1007.

5. Parrish JJ, Susko-Parrish JL, Winer MA, et al. Capacitation of bovine sperm by heparin. Biol. Reprod. 1988;38:1171.

6. Palmer E, Duchamp G, B6zard J, et al. Non-surgical recovery of follicular fluid and oocytes of mares. J. Reprod. Fert., Suppl. 1987;35:689.

7. Bruck I, Raun K, Synnestvedt B, et al. Follicle aspiration in the mare using atransvaginal ultrasound-guided technique. Equine vet. J. 1992;24:58-59.

8. Cook NL, Squires EL, Ray BS, et al. Transvaginal ultrason- ically guided follicular aspiration of equine oocytes. ,I. Equine vet. Sci. 1992; 12:204-207.

SPERM CONCENTRATION INFLUENCES RECOVERY OF PROGRESSIVELY MOTILE SPERMATOZOA AND

NUMBER OF INSEMINATIONS SHIPPED IN CONVENTIONAL CONTAINERS

G. W. Webb PhD 1, M. J. Ams PhD 2, and K. C. Pool, BS 3

SUMMARY

Three ejaculates from each of 4 stallions were split into aliquots and diluted with skim milk-glucose extender to final concentrations of 25, 50, 100, 150, or 200 million progressively motile spermatozoa/ml. One aliquot per dilution was evaluated for the percentage progressive motility (PMS) and rate of forward movement (RFM) after 15 and 45 minutes of culture at 37°C. One aliquot was stored at 5°C for 24 hours and another for 48 hours at 5°C. The aliquots stored at 5°C were evaluated for PMS and RFM at 15 and 45 minutes of warming (37°C). For spermatozoa stored at 37°C following collection, concentration did not (P<.05) influence

Authors addresses: 1Equine Sciences Program, Colby Community College, Colby, Kansas, 67701. 2Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas, 66506. ~Northeast Oklahoma A&M College, Miami, Oklahoma, 74354

PMS or RFM. However, when spermatozoa were stored at 5°C for 24 or 48 hours, PMS and RFM declined (P<.05) as spermatozoal concentration increased. A concentration of 25 x 106 progressively motile spermatozoa/ml provided higher (P<.05) percentages of PMS following storage than did concentrations of 100,150, and 200 x 106 progressively motile spermatozoa/nil. However, in order to minimize insemination volume and maximize the number of insem- inations available per shipment, a concentration of 100 x 106 progressively motile spermatozoa may be more appropriate than lower concentrations.

INTRODUCTION

Development of the technology to store and ship equine semen in the liquid form has resulted in increased marketing opportunities forsome breeders. In addition, on the farm use of this technology has increased the flexibility of collection schedules and made more efficient use of available labor. During the development of this technology research was, and continues to be, conducted on cooling rates, extenders, and dilution rates which will maximize the percent of spermatozoa that retain motility after storage. 1,a,a It has been reported that semen samples diluted to a final concentration of 25 x 106 spermatozoa/ml exhibit recovery rates superior to those diluted to higher concentrations. 3 However, when concentration is held constant, samples with less than 20 percent seminal plasma in the

488 JOURNAL OF EQUINE VETERINARY SCIENCE