Embryo production by ovum pick up from live donors

ELSEVIER

EMBRYO PRODUCTION BY OVUM PICK UP FROM LIVE DONORS C. Galli,’ G. Crotti, C. Notari, P. Turini, R. Duchi and G. Lazzari Laboratory of Reproductive Technologies (LTR) Consorzio per I’ Increment0 Zootecnico (CIZ) Via Porcellasco 7/F, 26100 Cremona, Italy Received for publication : August 26, 2000 Accepted : September 26, 2000

ABSTRACT Embryo production by in vitro techniques has increased steadily over the years. For cattle where this technology is more advanced and is applied more, the number of in vitro produced embryos transferred to final recipients was over 30,000 in 1998. An increasing proportion of in vitro produced embryos are coming from oocytes collected from live donors by ultrasound-guided follicular aspiration (ovum pick up, OPU). This procedure allows the repeated production of embryos from live donors of particular value and is a serious alternative to superovulation. Ovum pick up is a very flexible technique. It can be performed twice a week for many weeks without side effects on the donor’s reproductive career. The donor can be in almost any physiological status and still be suitable for oocyte recovery. A scanner with a sectorial or convex probe and a vacuum pump are required. Collection is performed with minimal stress to the donor. An average of 8, to 10 oocytes are collected per OPU with an average production of.2 transferable embryos. The laboratory production of embryos from such oocytes does not differ from that of oocytes harvested at slaughter as the results after transfer to final recipients. For other species such as buffalo and horses OPU has been attempted similarly to cattle and data will be presented and reviewed. For small ruminants, laparotomy or laparoscopy seems the only reliable route so far to collect oocytes from live donors. CD 2001 by Ekevier Science Inc. Key words: ovum pick up, cattle, buffalo, horse, embryo

Acknowledgments The authors acknowledge the technical support of Mr. Massimo lazzi, Dr. Fabio Ferrara and Dr. Stefano Allodi. Part of this work was supported by 2 EC grants CT9501 90 and CT98-0032. ’ Corresponding author Theriogenology 55:1341-1357,200l 6 2001 Eleevier Science Inc.

0093-691WOlB-see front matter Pit: SOOSS-691X(01)00466-1

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INTRODUCTION Embryo transfer technology has been applied extensively for cattle to increase the number of offspring that can be obtained from the very best donors. Since the early eighties superovulation has had a tremendous impact on cattle breeding. At present over 500,000 embryos per year are obtained worldwide by this technique and about 440,000 are transferred (64). With the development of safe and repeatable techniques of in vitro embryo production, a growing percentage of the embryos obtained from the best donors are produced in the laboratory. The number of in vitro produced and transferred embryos worldwide has now reached about 31,000 (64). For early in vitro procedures the source of oocytes was the ovaries of slaughtered donors (43,22, 24). Now the oocytes from valuable donors are more frequently recovered from the live animals by transvaginal ultrasound-guided oocyte collection (Ovum Pick Up, OPU) (52, 65). A particular case is represented by very young donors such as heifer calves of 2 to 3 months of age whose oocytes are collected surgically by follicular aspiration through laparotomy or endoscopy. In this paper we describe the fundamental steps of the in vitro embryo production procedure starting with the collection of oocytes from live donors. We focus on cattle, species in which this technology is most advanced and used; we also present original data for buffalo and we briefly review data on horse, sheep and goats.

POTENTIAL APPLICATION OF THE VITRO EMBRYO PRODUCTION TECHNIQUE FROM LIVE CATTLE DONORS In vitro embryo production from live donors is an extremely versatile technique because it can be applied to donors of all ages from two-months-old calves to very old cows. It can be used for dry and lactating donors and even during pregnancy up to the third or fourth month. It does not interfere with the physiological status of the donor since no hormonal stimulation is required. However, in certain instances, it can have a beneficial effect. It is normally applied in a regime of two ultrasound-guided oocyte collections per week. Thus in a very short time several batches of embryos fertilized with the desired bulls can be obtained. Different schedules can be used for oocyte recovery allowing the system to adapt to different practical situations. Twice-a-week collection (usually Monday and Thursday) allows the maximum recovery of oocytes of suitable quality for embryo production in a given time interval. Once-a-week collection results in the recovery of a smaller number of oocytes (of lower quality) that have already undergone cumulus expansion and atresia (our results, 25). This is because in the twice-a-week collection, if all visible follicles are aspirated no dominant follicle develops. In most once-a-week collections a dominant follicle is present at each successive collection that causes the regression and degeneration of the subordinate follicles. In the once-a-week collection schedule the donors also can come into estrus while this is never the case in the twice-a-week schedule. Some authors prefer to combine superovulation with OPU and

1343

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to recover the oocytes before the onset of estrus (7, 28) but the procedure can be repeated at best every two weeks. The average yield of embryos can be higher per OPU but the total number over a period of time is lower than for the twice-a-week collection. OPU can have its own therapeutic effect on infertile donors, especially those affected by ovarian cysts. Most cow donors treated at our center have infertility secondary to repeated superovulation treatment. This is why we do not use gonadotrophin stimulation in our program. Virtually all empty donors come on cycle within two weeks from the last oocyte collection and can be inseminated successfully. The OPU has no side effect even after twice-a-week collections for over a year, as it has been the case for a few of our donors. In some cases though, a minor hardening of the surface of the ovaries can occur after several months of repeated collections. OPU is a much more demanding technique than superovulation both because of the more expensive and diverse equipment needed and the technical skills required especially for the laboratory procedures.

TYPE OF DONORS Virtually all female cattle starting from 2 months of age can be oocyte donors by follicular aspiration. The only exception are pregnant animals after the third or fourth month of pregnancy and animals with severe ovarian hypoplasia or in the immediate post partum before ovarian activity is restored. Calves of about 2 to 3 months can be oocyte donors. But for an efficient collection without side effects the procedure requires a laparotomy under general anesthesia. The ovaries are exposed through the incision and the oocytes are recovered by follicular aspiration. Young calves are ideal donors from the perspective of cattle breeding companies because the use of prepubertal animals allows them to reduce the interval between generations and therefore accelerates the process of genetic improvement (40). For this type of donor a number of investigators have demonstrated the need for a treatment of hormonal stimulation to obtain oocytes with higher developmental capacity. The question of the most suitable protocol of ovarian stimulation for collecting the highest number of competent oocytes has been addressed by several laboratories (1, 2, 12,31, 38,41,58, 80). Differences in protocols for ovarian stimulation and in the type of donors (breed, age) are probably responsible for the controversial data reported in the literature: development up to the blastocyst stage has been found to be either higher (1), equal (2, 31) or lower (12, 37, 42, 58, 83) compared to that of adult donors. Also, the viability of embryos obtained from calves has been found normal in some studies (1,38) and low in others (56). In our experience the most suitable protocol for Holstein Friesian calves is based on steroids and eCG (Table 1).

8

8

37

15

38

59

Calves 2-3 months of age (untreated)

Calves 2-3 months of age (eCG treated*)

Calves 6-8 months of age (untreated)

Heifers from 9 months of age (untreated)

cows (untreated)

1661 (6.4)

(5.9)

(9.9)

(9.9)

1271

2288

2579

485 (5.9)

595 (16.1)

Cleaved (per OPU) n.

795 (9.7)

1005 (27.2)

128 (16.0)

Oocytes (per OPU) n.

64.4

55.6

61 .O

59.2

50.0

Cleavage %

(2.9)

532

;z)

$7)

(E2)

$5)

Freezable embryos (per OPU) n.

655 (2.5)

(Z)

(E)

116 (3-l)

(067)

Transferrable embryos (per OPU) n.

32.0(d)

23.4 (c)

12.8 (b)

13.6 (b)

4.5(a)

Freezable embryos/ cleaved %

39.4 (d)

30.5 (c)

17.5 (b)

19.5 (b)

8.2(a)

Transf. embryos/ cleaved %

‘The protocol of ovarian simulation for 2-3 months eCG treated cakes was as follows: D+O: 5 mg estmcfiolvalerate and intravaginal progestagen releasing sponges (80 mg fluorogestone acetate), D+3 and D+i 1: 400 IU eCG, D+16: 1000 IU eCG, D+18: laparotomy and oocyte collection. Student T test. Values within columns with different letters are statistically different (P c 0.05). Galli and Lazzari, unpublished.

261

254

82

37

OPUS n.

Donors n.

Type of donor

Table 1. Developmental capacity of oocvtes collected from donors of different age with or without hormonal prestimulation. E

Theriogenology

For older donors (from 5 months onward), there are conflicting reports on the effect of pretreatment with gonodotrophins on oocyte collection and embryo development. Some authors have shown a beneficial effect (51) even on pregnant donors (47) while others have not found any difference compared with controls (17, 18). Other authors have shown an age-dependent mechanism whereby hormonal stimulation is beneficial only in young donors up to 3 months (Galli and Lazzari unpublished, see Table 1) or up to 9 to 10 months (53). Despite these conflicting data on the effect of hormonal pretreatment, several studies demonstrated (59) that the developmental capacity of the oocytes increases with the age of the donor, puberty being the relevant turning point.

PROCEDURE AND EQUIPMENT FOR TRANSVAGINAL ULTRASOUND-GUIDED OOCYTE COLLECTION (OVUM PICK UP) OPU is usually performed in residential centers where the environment can be better controlled for temperature, air contamination, and ease of work. Several companies also operate on farms if required by the customer. For the collection procedure the donor must be contained in a chute and usually is sedated with detomidine hydrochloride (DomosedanQ Vetem, Italy). In addition, an epidural anesthesia is always administered to facilitate the handling of the ovaries through the rectum. An ultrasound and aspiration equipment are needed for oocyte recovery from live donors. A number of different scanners and probes are suitable. In the literature the following equipment is mentioned: Aloka 500 with a 5 MHz convex array transducer housed in a Delrin plastic vaginal probe with stainless steel needle guide (42,29); Hitachi EUB 415 with a 8.5 MHz vaginal curved array transducer with a custom-made holder and a single needle guide (34); Aloka 500 with a 5 MHz curvilinear human transvaginal probe carrying a stainless steel needle guide (53); Capasee Toshiba with a 6 MHz human endovaginal probe with a custom-made holder (17, Galli and Lazzari, unpublished); Medison Sonovet 600 with an ad hoc endovaginal probe (Galli and Lazzari, unpublished); Hitachi EUB 405 with a 6.5 MHz finger-tip probe inserted into a custom-made holder (45); Pie Medical scanner 200 Vet with mechanical multiangle probe housed in a holder that allows the use of disposable hypodermic needles (4). If OPU is performed on a farm a portable scanner must be used (Aloka, Medison, Pie Medical). The gauge of the needle varies from 20G to 17G and the length from to 4 cm to 60 cm; the vacuum connected to the needle is regulated from 40 to 115 mm Hg (flow rate 20 to 25 mUmin) (5). We use a Toshiba Capasee (Toshiba Italia, Milano, Italy) and a Sonovet 600 (Medison, Seoul, Korea), with a custom-made 17G 55 cm single-lumen needle with a 19G 7-cm-long spinal cannula (Terumo Europe, Leuven, Belgium) mounted at the tip that is replaced every four OPU. Both probes have interchangeable stainless steel needle guides that are changed for every donor together with the needle. The choice of the scanner and probe also depends on the type of animal. In general the endovaginal probes are suitable for all donors. Probes adapted for endovaginal use by inserting them in a

1346

custom-made holder are too big for the vagina of young donors. The follicular aspirate is collected in a 16 mL Falcon test tube that is kept warm in a heating block (V-FTH1000, Cook ltalia srl, Milano). The aspiration is performed with a foot-operated pump (V-MAR-5000, Cook ltalia srl, Milano) set at 115 mmHg that with our tubing length results in a flow rate of 20 to 25 mL /minute). With this setting we estimate a oocyte recovery rate of 55 to 60% of the follicles punctured. This setting is a compromise between a good recovery rate and minimal damage of the cumulus oocyte complex. In our experience a higher aspiration pressure increases the total number of oocytes recovered but also increases the percentage of denuded or damaged oocytes. The follicular aspirate always contains blood and the oocytes are immediately searched with the aid of embryo specific filters (i.e., Emcon); in our laboratory we use disposable cell strainers with 70 urn pores (Falcon, cat.n’ 2350, Becton Dickinson, Milano, Italy).

IN VITRO MATURATION The medium used most for in vitro maturation of cattle oocytes is TCM 199 (Tissue Culture Medium 199) bicarbonate buffered. Supplements include 10% Fetal Calf Serum, FSH, LH, estradiol and EGF (45,48). In our experience, the developmental capacity of the oocytes improves significantly if additional granulosa cells (23) or exogenous growth factors such as IGF I and EGF (19) or heparin (39) are added to the maturation medium. Maturation time varies between 22 and 24 h. However, if necessary for practical reasons, fertilization can start anytime from 18 to 26 h post IVM (in vitro maturation). This flexibility is necessary when many donors have to be fertilized with several different bulls, in order to concentrate the IVF procedure in a relatively short period of time. Maturation can take place in conventional CO, incubators in petri dishes, in four-well dishes or in microdrops covered with oil. In our laboratory the oocytes are matured in petri dishes or in fourwell dishes on a non-static platform inside the incubators. Alternatively maturation can occur in sealed plastic tubes containing CO,-equilibrated IVM medium, in batterypowered portable incubators even during transport from the location of collection to the laboratory. A number of companies now operate on this basis for logistical reasons to access donors located away from the embryo production center.

IN VITRO FERTILIZATION In large-scale commercial OPU embryo production programs the bull semen to be used is pre-tested on oocytes collected from slaughterhouse ovaries. In this way the optimal concentration of semen for achieving high monospermic fertilization is determined for each bull (29; Sybrand Merton, personal communication). Medium TALP (Tyrode Albumin Lactate Pyruvate) is generally used, supplemented with heparin and penicillamine, hypotaurine and epinephrine (PHE) in 5% CO? and 20% 0,. We have used these conditions for several years (24). Recently we demonstrated that both the level of oxygen during IVF and the addition of amino acids to the IVF medium can have a positive effect on fertilization rates and on development. Our data show

Theriogenology

1347

that lowering the oxygen level to 5% during IVF improves the fertilizing ability of bovine spermatozoa. For most bulls the optimal sperm concentration for achieving high fertilization is lower in 5% oxygen than it is in 20% oxigen. For some bulls that do not fertilize in 20% oxygen a high fertilization rate can be obtained in 5% 0, (36). We further improved the IVF conditions by switching from medium TALP to medium SOF (Synthetic Oviductal Fluid) and by adding amino acids (non essential amino acids NEAA, essential amino acids EAA). We compared the effects of different IVF media (TALP, SOF, SOF+NEAA, SOF+EAA+NEAA) on cleavage rate and development. Our results indicate that the quality of embryos fertilized in medium SOF supplemented with amino acids is superior because it is higher the percentage of freezable blastocysts (Table 2) (37). Table 2. Effect of different media on cleavage rate and embryo development of bovine oocvtes Tot.blast. Tot.blast. IVF medium Oocytes Cleaved Cleavage Freezblast. n. n. D+7fSEM D+7fSEM D+8fSEM TALP

218

188

78.4% a

27.8k5.2 a

19.7f4.5 a

40.9k3.8 a

SOF

207

171

80.9% a

29.7f4.7 a

19.8flO.4 a

37.5f4.5 a

SOF+NEAA

208

155

73.7% a

29.9k8.2 a

22.7f7.8 a

45.2f4.9 a

SOF+E+NEAA

197

153

78.2% a

35.7rt5.1 a

30.5i5.2 b

48.5f3.8 a

Student T test. Values within columns wih different letters are statistically different (P < 0.05).

At present, in our laboratory each bull is pretested on abattoir oocytes at a concentration of 0.5 million spermatozoa per mL and 1 ug /mL of heparin in SOF medium supplemented with amino acids in 5% CO, and 5% 0,. Working concentrations for optimal fertilization rate vary between 0.1 and 2 million sperm per mL. The semen is generally separated on a 45-90% Percoll gradient to ensure maximum recovery of the motile fraction; on average half or a third’of a straw of semen is sufficient for IVF.

CULTURE OF FERTILIZED OOCYTES After IVF, the presumptive zygotes are transferred to an in vivo or in vitro culture system for further development. In our OPU program we used primarily the in vivo culture system in which fertilized oocytes that have cleaved in vitro in microdrops of SOF are transferred to the ligated oviducts of sheep (temporary recipients) and developed to day seven. The comprehensive data of our OPU program referring to the past 3 years and the first four months of the year 2000 are shown in Table 3.

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Table 3. Ovum piok UD activity at the Laboratow of Reproductive Technologies OPUS occvtes Freezable Year Donors OPUS on heifers (per bPU) Cleavage embryos(Gl+GZ) n. n. donors n. % (per OPU) n. 1% of cleaved]

Transferrable embryos (per OPU) [% of cleaved]

1997

194

983

60.8

8300 (8.4)

60.3

1312 (1.3) [26.2]

1845 (1.7) [32.8]

1998

199

1266

56.2

10402 (8.2)

70.8

2192 (1.7) [29.7]

2619 (2.1) [35.5]

1999

134

1071

34.8

11041 (10.3)

69.1

2457 (2.3) [32.2]

3106 (2.9) [40.7]

2000 (4 months)

60

345

41 .o

3259 (9.4)

72.4

823 (2.4) KM.91

1022 (3.0) 143.3)

Other laboratories have used coculture systems in medium 199 or Menezo B2 with various cell types: oviductal cells (7) Buffalo Rat Liver cells (29, 67), Vero cells (lo), granulosa cells (8, 26, 49) or conditioned medium (14, 66). Cell-free systems with various modification of SOF medium (63,68) or CR1 medium (57). In recent years most coculture systems were abandoned in favor of cell-free systems at a low oxygen level. In our experience, in vitro culture systems have improved dramatically in the last few years. In our laboratory, although most embryos are developed in vivo in the sheep oviduct, a growing percentage of the embryos produced for commercial purposes are grown totally in vitro in SOF-BSA-AA medium. Under these in vitro culture conditions we addressed the question of the effect of the number of embryos cultured per 20 pL drop on blastocyst development. For this purpose we analyzed the data on a single group of donors of a beef breed, splitting the OPU collections according to the number of cleaved embryos per collection: 4 or less vs more than 4. In the two groups the average number cleaved was 2.8 (11 OPUS) and 7.9 (32 OPUS) and the percentage of freezable blastocysts on the cleaved was 61.3% and 44.8% respectively. In the low-number group in two collections we had 1 cleaved embryo each; both these single embryos developed in freezable blastocysts. These data indicate that the number of OPU embryos cultured per drop does not seem to affect further development. It is difficult to compare our data with other literature referring to large scale commercial OPU programs because the variables differ greatly. The most important source of variability is the type of donor followed by the different protocols used in the laboratory. In three programs in which all donors were problem cows the average number of oocytes collected was 4.9 (29) 6.3 (42) and 9.5 (7); in another program where donors were pregnant heifers and first-parity cows, the average number of oocytes per OPU was 7.6 to 7.8 (67). The average number of transferable embryos in these programs was 0.9 (15.4% of cleaved) (42), 4.7 (63.1% of cleaved) (7) and 1.l to 1.4 (14 to 17% of cleaved) (67-68).

Theriogenology

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SEXING OF OPU EMBRYOS Embryos produced by OPU can be sexed by biopsy and DNA amplification by PCR (exactly as embryos produced conventionally by superovulation). In our experience the pregnancy rate of sexed and frozen embryos has been remarkably similar to unsexed ones. We perform the biopsy only on grade 1 embryos (always in the laboratory) with a Leitz mechanical micromanipulator. The micromanipulator allows us to remove (with a blade) a very low number (2 to 8) of cells thus reducing the damage. All the embryos are frozen shortly after biopsy without waiting for DNA analysis. Perhaps the breaching of the zona helps in establishing the pregnancy. In our OPU program (Table 4) we sexed a total of 1920 embryos. The total percentage of females was 43.7 but it varied from 40 to 47% at different times. Tab. 4. Sexing of OPU embrvos at the Laboratory of Reproductive Technoloales Year Males Undetermined Females Embryos Females n. % sexed n. n. n.

Undetermined %

1997

203

67

106

10

46.1%

4.9%

1996

653

296

336

19

47.0%

2.9%

1999

641

327

490

24

40.0%

2.9%

2000 (4 months)

223

96

113

12

46.4%

5.3%

Total

1920

610

1045

65

43.7%

3.4%

A similar sex distribution is reported by others (45.2 to 49% referred to female calves born, 29) while in another study the percentage was lower (37.5%, 7). These results are somehow different from the expected sex ratio of 47 to 49% female calves derived from in vivo embryo transfer or Al (15, 32). However, in a recent study carried out from July 1997 to March 2000 on 1395 embryos produced in vivo by superovulation and 558 produced in vitro the sex distribution was very similar between the two groups (in vivo: 51.3% females; in vitro 49.1% females) and not different from the above cited data on Al (Tom Otter, personal communication).

PREGNANCY RATE OF OPU EMBRYOS In our experience the pregnancy rate of in vitro produced embryos grown in the sheep oviduct is not different from that of in vivo produced embryos even after freezing and thawing. Table 5 shows the data collected in our nucleus herd where we compared the pregnancy rate of frozen-thawed embryos produced either in vitro or by superovulation. The data are shown separately for embryos frozen in glycerol or in ethylene glycol.

1350

Theriogenology

Table 5. Preonancv rate of in vitro produced and in vivo produced embrvos in Cli! nucleus herd In vitro embryos (OPU) In vivo embryos Cryoprotectant n.transferred/n. pregnancies n.transferredln. pregnancies (preanancv rate) (preanancv rate) 10% glycerol

92/l 66 (55.4%)

212/399 (53.1%)

1.4 M ethylene glycol (direct transfer)

36l76 (47.4%)

62/l 06 (57.4%)

Data for sexed embryos (not shown) do not differ from unsexed embryos. In summary, we did not find any difference between in vitro and in vivo embryos or between cryoprotectants (glycerol vi&vitro 53.1 o//55.4%; ethylene glycol viva/vitro: 57.4%/47.4%). Other data are reported in the literature for frozen-thawed Grade 1 embryos totally grown in vitro and frozen in 10% glycerol (68). In that study 575 embryos grown in two different in vitro culture systems were transferred; the pregnancy rate was 38.8% for the group developed in a cell-free system based on medium SOF, and 42.1% for the group developed in a coculture system based on BRL cells. In an earlier study, in which a direct comparison was made between in vivo and in vitro embryos grown in coculture with BRL cells, the pregnancy rate of vitro embryos both fresh and frozen was significantly lower than that of in vivo embryos (fresh viva/vitro: 76%/59%; frozen vivohritro: 67%/42%, 29). This is in contrast with our data in which we obtained the same pregnancy rate transferring in vivo and in vitro produced embryos. It must be noted though, that all our in vitro embryos were grown in the sheep oviduct from Day 2 to Day 7 and this difference may be responsible for their high viability even after freezing and thawing. After pregnancy diagnosis there is a physiological expected incidence of abortion. In one study of 10 Holstein dairy herds (ranging from 130 to 700 cows each) the abortion rate after Al was between 6.3 and 13% (16). A comparison between losses after Al (0.5 to 1.3%), in vivo embryo transfer (1 .l to 5.9%) and IVP (1.3 to 6%) was published recently (68). In this study a significant difference was found between IVP and Al in the largest field trial but not in two smaller trials. Our own data indicate pregnancy losses of 7.3% for in vivo embryo transfer and 11.1% for IVP.

CALVING DATA FROM OPU EMBRYOS Data have been published on the characteristics of calves derived from in vitro produced embryos compared to calves derived from Al or superovulation (MOET) (29, 35,68). These studies indicate that IVP calves have a higher birth weight, longer gestation, higher percentage of male calves, congenital malformations, cesarian sections and perinatal mortality than Al calves (and often MOET calves). However, one field trial (68) indicated that these negative effects can be reduced by using a cell-

Theriogenology

1351

free system based on SOF medium instead of a coculture system with BRL cells. The type of culture system has a clear effect on the developmental capacity of the oocytes. These studies demonstrate that this effect is long lasting. Our own limited data on calving within our nucleus herd also indicates slight differences in gestation length between in vivo embryo transfer and IVP (277 versus 278.7 days) and in the incidence of perinatal death (8.8 versus 7.7%).

OPU IN OTHER SPECIES Buff alo Most of the work on buffalo has been with slaughterhouse ovaries (44). Recently ovum pick up has been extended to this species as well (8, 33). In our laboratory we have worked with lactating and repeat breeder non-lactating females using exactly the same procedure that we use on cattle both for OPU and for embryo production; interestingly we observed that the buffalo embryos are12 to 24 h more advanced than the bovine counterpart developing in parallel. Five donors were subjected to OPU for several weeks and the number of oocytes recovered remained constant throughout (unpublished data). The results we obtained (Table 8) demonstrate that OPU is even more competitive in buffalo than in cattle when compared to superovulation. Table 6. Embrvo production bv ovum pick up in buffalo Donors n.

5

OPUS n.

67

(per OPU) n.

Cleaved (per OPU) n.

Cleavage %

306

125

46.1%

Oocytes

Freezable embryos (per OPU) n.

Freezable embryos/ cleaved % 36.4%

(4.5)

Nine frozen thawed embryos were transferred to synchronised recipient heifers and resulted in the birth of 3 buffalo calves (21). Buffalo frozen semen has been the main source of variation and the different bulls used for in vitro fertilization showed a high degree of variation with an average cleavage rate of only around 40%. Recently the cleavage rate has been higher suggesting that the quality of the frozen semen has improved in general and that fertility is not dependent on individual bulls. With this degree of efficiency OPU can be considered seriously as an efficient tool for genetic improvement of buffalo.

1352

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Horse Oocyte recovery from live mares is complicated by anatomical and physiological differences when compared to large ruminants (30). The collection of immature oocytes is performed transvaginally as in cattle. However it is necessary to flush repeatedly the follicle with medium to increase the chances of recovering the oocyte. The recovery rate is quite low and might range from 18 to 35% with an average of 2.4 oocytes per OPU (9). More successful is the recovery of oocytes from the preovulatory follicle just before ovulation (50). In vitro matured oocytes transferred to the oviduct of inseminated recipients can develop to blastocysts at a very high rate (69). The procedures for in vitro maturation are identical to those for cattle, with the difference that compact cumulus oocyte complexes take a few hours more to reach metaphase II (about 26 to 28h). In vitro fertilization is not efficient and only a limited number of foals have been obtained after this procedure (50). lntracytoplasmic sperm injection (ICSI) is a likely answer to the IVF problem in horses (11). The ICSI of in vivo matured oocytes transferred back to the oviduct of recipient mares has resulted in the birth of foals (46). In our laboratory we have performed ICSI with slaughterhouse oocytes matured in vitro. We achieved cleavage (74%) and blastocyst (24%) rates similar to those for the cattle in vitro system (20). The embryos obtained were frozen and are currently being transferred. The developmental competence of oocytes with an expanded cumulus at the time of collection was only about a third of that of oocytes with a compact cumulus at recovery. Culture requirements of horse oocytes look similar to those of ruminants. In fact after cleavage they were embedded in agar chips and transferred to sheep oviduct for in vivo culture. The use of this technology in mares is foreseen as a therapy for infertility rather then a system to increase the number of offspring from a given mare. Sheep and Goats The collection of oocytes from live ewes and does is performed either laparoscopically (3, 58, 62) or surgically by laparotomy (55). It has also been described for does, using transvaginal ultrasound-guided follicular aspiration (27). In small ruminants most authors use a stimulation protocol with gonadotrophins to increase the size of the follicles and to facilitate the puncture. If endoscopic or surgical recoveries are repeated several times on the same donor they can cause adhesions that might compromise the subsequent fertility of the donors. Large numbers of oocytes (30 to 60) can be collected per session from lambs a few weeks old (13, 55). In adult donors the number of oocytes collected per OPU is similar to that for cattle and can be in the range of 6 to 8 for sheep and goats. The efficiency of the embryo production system is also comparable to that of cattle and each OPU session in adult donors can result in the production of 1 to 3 transferable embryos using identical culture conditions. The lambing rate of fresh OPU-produced embryos in sheep was 41% (54).

1353

Theriogenology

CONCLUSIONS Oocyte recovery from live donors is an important source for the production of embryos from individual donors of particular value and for particular scopes. For large animals ultrasound-guided follicular aspiration is the technique of choice for its simplicity, non-invasiveness, repeatability and efficiency. For small ruminants, more complex approaches such as laparotomy or laparoscopy are required. This makes the procedure more invasive and not repeatable in the long term because of possible side effects. The subsequent production of embryos (IVM, IVF, IVC) is independent of the source of oocytes. In our experience the rates of development are similar or higher when the oocytes are recovered from live donors as compared to oocytes originated from the ovaries of slaughtered animals. Currently, commercial application is significant only for cattle. Thus, OPU could be an important tool for the genetic improvement of buffalo and for assisted reproduction of infertile mares. The cost of the technology is justified when an individual animal is valuable.

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