Th.A.M. Kruip

In vitro produced and cloned embryos: Effects on pregnancy, parturition and offspring

Earlier reports have indicated that the transfers of bovine and ovine embryos produced by in vitro procedures (IVP) or by nuclear transfer (NT) have resulted in the birth of heavy offspring. The present paper presents summary information from 30 data sets obtained worldwide (WW) on IVP and NT in different cattle breeds, plus the preliminary results from a highly controlled field study (FS) undertaken by Holland Genetics (HG) on Holstein-Friesian IVP calves. Data of artificial insemination (AI) and embryo transfer (ET) served as controls. The results from the WW and FS trials were very similar. After adjusting for such effects as season, parity of cow/recipient and sex of calf, foetal losses between pregnancy diagnosis and term were higher for IVP and NT embryos. In addition, both gestation length and birth weight were increased relative to AI and ET calves, and there were also higher incidences of Dystocia, perinatal loss and anomalies. The increased incidence of these problems has important implications for animal health and welfare, as well as affecting the commercial acceptance of these techniques. Research should focus on each step in IVP and NT procedures and on the synchrony between embryo and uterus at transfer, to understand and overcome these problems.

Aspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries

A technique for the repeated collection of bovine oocytes using transvaginal ultrasound guided aspiration is described. Cows were used during their normal estrous cycle and after stimulation of the ovaries with pregnant mare serum gonadotrophin (PMSG). The sedation of the animals and the puncturing of follicles appears not to have traumatized the animals and plasma progesterone measurements suggested that the cyclicity was not interrupted. A total of 36 transvaginal aspiration procedures were performed, during which 54 oocytes were recovered from 197 follicles. These experiments indicate that the repeated aspiration of bovine oocytes during transvaginal ultrasound scanning is possible. However, more research is needed to establish optimal methods for improving the recovery rate of oocytes before this method can be used as an alternative route for the supply of oocytes for in vitro maturation and in vitro fertilization.

TRANSVAGINAL ULTRASOUND GUIDED FOLLICULAR ASPIRATION OF BOVINE OOCYTES

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.

Potential use of ovum pick-up for embryo production and breeding in cattle

The efficacy of transvaginal ultrasound-guided puncturing of ovarian follicles for collecting immature oocytes in cattle was studied. Three experiments were conducted to examine the effects of puncturing on follicle recruitment and on the number of oocytes collected. Puncture sessions were executed twice weekly at regular intervals of 3 and 4 d respectively. The oocytes were matured, fertilized and allowed to develop in vitro and the number of transferable embryos was recorded. The health of the cows was checked daily. In Experiment 1, dairy cows (n=10) were punctured over a period of 5 mo, and the collected oocytes were fertilized with the semen of 1 bull. In Experiment 2, oocytes were collected from one 12 year old high pedigree dairy cow and an one month pregnant cow were punctured. The oocytes of the old cow were fertilized with semen of 8 different bulls. In Experiment 3, beef cows (n=6) were punctured over a 2 mo period and the semen of 2 different bulls of the same breed was used to fertilize the oocytes from 3 of these cows. In Experiment 1, 14.5 +/- 0.4 (mean +/- SEM) follicles were punctured per session, and 8.0 +/- 0.3 (mean +/- SEM) oocytes were recovered. A mean of 16% of the oocytes developed into transferable embryos with a pregnancy rate of 40%. The results did not differ between the months of the experiments, indicating that the transvaginal puncturing method can be used successfully over a 5 mo period. No detrimental effects were observed after clinical and post mortem examinations, nor did breed, age or reproductive status appear to affect the results. However, large differences were observed between individual cows and between cow/bull combinations.

Follicular dynamics, repeatability and predictability of follicular recruitment in cows undergoing repeated follicular puncture

The aim of our study was to characterize follicular recruitment and growth in response to the repeated removal of follicles. All tertiary follicles (> 2 mm of diameter) in the ovaries of 10 non-lactating Holstein Friesian cows were punctured at midcycle (Day 0) by means of an ultrasound-guided needle. Puncture sessions were scheduled twice weekly at 3- or 4-d intervals over 3 mo. In the middle of the experiment, i.e., Week 7, the effects of 2- and 5-d intervals between follicular punctures were tested and compared with the previous 3- and 4-d intervals. After this period, 6 animals were slaughtered to study the effect of puncturing on gross ovarian morphology. The protocol of puncturing follicles with 3- to 4-d intervals was continued for an additional 3 mo in the remaining 4 animals. Twice-weekly puncturing of all tertiary follicles abolished estrous cycles and lead to an increase in follicular wave frequency without apparent negative effects on either the reproductive tract or ovaries. After puncture the new follicular wave attained full numerical development within 3 d. Two-day intervals resulted in a lower number of follicles than the 3-d interval (11.0 -/+ 3.8 vs 15.4 -/+ 6.1; P < 0.05). In contrast 4- and 5-d intervals between puncture resulted in an increase in follicle size when compared with that of the 3-d interval. The mean+SD number of recruited follicles varied between animals ranging between 78 +/- 2.5 to 19.2 -/+ 6.0. The mean number of follicles recruited increased from the first month (March) to the third month (May) of sampling (11.8 +/- 4.7 vs 16.4 +/- 6.5; P < 0.01), and then decreased between the third (May) and the sixth (August) month of sampling (21.5 +/- 4.7 vs 16.8 +/- 5.0; P < 0.01). During the experiment, the number of recruited follicles varied cyclically, with waves having a length of 6 puncture session (PS) or 3 wk (i.e., the mean length the bovine estrous cycle). Follicular recruitment after repeated ovum pick-up showed a high repeatability (r = 0.576) A model was also developed showing good predictability of the potential of animals to recruit follicles on the basis of the first 4 to 6 PS. Our results showed that despite large variation in follicular recruitment between animals, the high repeatability and good predictability of follicle recruitment demonstrates the possibility of characterizing animals on their potential for follicle growth.

Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and stage of the oestrus cycle

Eight hundred and seven bovine antral follicles from 2 mm to 20 mm in diameter were dissected free of stromal tissue, measured, qualified and divided into 36 groups according to size, quality and stage of cycle. The follicular fluid was collected and assayed by RIA for oestradiol-17beta, testosterone and progesterone. The steroid hormone concentrations vary with follicular size, degree of atresia and stage of the cylce. Non-atretic follicles of less than 8 mm are generally androgen-dominated and non-atretic follicles of more than 11 mm are oestrogen-dominated. Follicles betwen 8 mm and 11 mm are intermediate in this respect. Degeneration leads to a gradual decrease of oestradiol-17beta and testosterone concentration and increase of progesterone. It is suggested that the ratio of oestradiol-17beta/testosterone and oestradiol- 17beta/progesterone and oestradiol-17beta/testosterone + progesterone cannot generally be used to discriminate between non-atretic and atretic follicles. Large follicles present during the early luteal stage contain as much oestradiol-17beta in the follicular fluid as large follicles during the follicular stage, whereas large follicles of the luteal stage contain only 15% of the maximal amount of the latters. This and other presented data support the statement that follicles present during the early luteal, late luteal and follicular stages of the cycle belong to different groups of growing follicles. It has been concluded that groups of macroscopically qualified follicles can be distinguished from each other by the steroid hormone concentration in the follicular fluid. It is therefore possible to predict the hormonal environment of the oocyte in any individual follicle of a defined size and quality.

Follicular and oocyte maturation in cows treated for superovulation

The maturational stage of oocytes and their follicles was assessed at 24/26 h after the preovulatory luteinizing hormone (LH) peak by means of morphological criteria. Follicles were obtained from cows treated for superovulation (PMSG/PG) with additional anti-PMSG to neutralize the residual PMSG. Follicular fluid was also recovered and analyzed for progesterone and estradiol levels. Seventy-two percent of the oocytes were at the Metaphase II (M(II)) stage of meiosis, whereas only 28% of the follicular walls were at the proper maturational stage; assessed on morphological characteristics, 78% of the follicles were progesterone-dominated. Earlier maturational stages of oocytes and follicles were also present, including those that are restricted to periods shortly after the LH peak in the normally cyclic cow. It is concluded that upon treatment for superovulation not all oocytes and follicles mature synchronously, and that not all oocytes mature in harmony with their follicles.

Characteristics of bovine estrous cycles during repeated transvaginal, ultrasound-guided puncturing of follicles for ovum pick-up.

Repeated transvaginal ultrasound guided puncturing of visible follicles was performed for ovum pick-up (OPU) during Periods A and B, each of which lasted 3 mo. During Period A, 10 cows (A) were used in the study. Period B commenced 1 mo after Period A and two groups of animals were used. The first group (B1) consisted of 9 of 10 cows from Group A. The second experimental group of animals in Period B consisted of 11 cows (B2) which had not been submitted to previous puncture. During the study, all visible follicles larger than 3 mm were punctured and aspirated three times, on Day 3 or 4, Day 9 or 10 and Day 15 or 16 of the estrous cycle. The mean estrous cycle length (+/- SEM) after repeated follicle puncture did not differ among the three groups and was 22.3 +/- 0.4, 22.5 +/- 0.4 and 22.1 +/- 0.3 d for groups A, B1 and B2, respectively. The mean total number (+/- SEM) of punctured follicles per estrous cycle in Group A (13.1 +/- 0.5) was significantly larger than in Groups B1 (11.2 +/- 0.4) and B2 (11.6 +/- 0.4). The largest number of follicles punctured for ovum pick-up in all three groups was always on Day 3 or 4 of the estrous cycle: 4.9 +/- 0.3 follicles; the mean (+/- SEM) number of punctured follicles on Day 9 or 10 and Day 15 or 16 was significantly (P<0.05) lower: 3.4 +/- 0.2 and 3.9 +/- 0.2, respectively. In Period A, primarily 3- to 5-mm follicles were punctured per estrous cycle, while 6- to 10-mm follicles were predominantly punctured in Period B (P<0.05). Recovery rate of oocytes on Day 3 or 4, Day 9 or 10 and Day 15 or 16 were 53, 50 and 52%, respectively. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm.

Bovine cumulus-oocyte-complex-quality is reflected in sensitivity for α-amanitin, oocyte-diameter and developmental capacity

The aim of the present study was to find more parameters to define developmental competence of cumulus-oocyte-complexes (COCs). Bovine COCs were divided into five groups based on their morphology. In order of increasing level of atresia: COC-A had a bright, compact cumulus investment; COC-B1 also had a compact cumulus investment, but was darker than COC-A; the color of COC-B2 was comparable with COC-B1 but the corona radiata appeared to dissociate from the rest of the cumulus investment; the cumulus of COC-B3 was almost black and the corona radiata was almost completely dissociated from the rest of the cumulus investment; COC-C had a strongly expanded cumulus investment with dark spots of degenerated cells. An increasing level of atresia was accompanied by: (1) an increasing zona pellucida diameter (147.6, 150.8, 151.0, 154.3 and 155.1 microm, respectively, for COC-A, COC-B1, COC-B2, COC-B3 and COC-C); (2) an increasing oocyte diameter except for COC-C (120.9, 122.8, 122.8, 123.9 and 118.4 microm, respectively); (3) an increasing developmental competence except for COC-C (13.9, 14.7, 17.4, 19.1 and 11.5%, respectively, development to morula and blastocyst after in vitro embryo production (IVP)) and (4) by a increasing percentage of oocytes exhibiting germinal vesicle breakdown (GVBD) after 24h culture with alpha-amanitin and FSH, except for COC-A (34.4, 68.6, 51.0, 22.0 and 4.8%, respectively, oocytes arrested in GV). In general, embryo-quality, expressed in nuclei-count, was significantly affected by COC-quality (P < 0.05) with B3 > B1> B2 > A > C. However, if the developmental stages were compared separately, this effect was less evident. It was found that, in absence of FSH, alpha-amanitin was unable to inhibit GVBD and that the success of GVBD inhibition was positively correlated to the amount of cumulus cells surrounding the oocyte. COCs that had been exposed to alpha-amanitin and FSH during maturation retained the ability to cleave after IVF, but were unable to develop any further.

Developmental potential of in vitro produced bovine embryos following cryopreservation and single-embryo transfer

Abstract Information on the developmental potential of in vitro-produced, cryopreserved bovine embryos following single-embryo transfer to synchronized recipients is limited. The aim of this study was to compare the pregnancy rates of vitrified, conventionally cryopreserved, and control (unfrozen) in vitro embryos. The embryos were cryopreserved either by Vitrification in Solution 3a (VS3a) or by conventional slow freezing in 10% glycerol. Forty-two percent (n=121) of the recipients established pregnancy when in vitro produced morulae to expanded blastocyst stage embryos were transferred immediately following culture. Singnificantly more morulae on Day 7 and 8 of culture established pregnancy ( 11 14 ) than blastocysts on Days 7 to 9 of culture ( 40 107 ; P 12 35 ) than those on Days 7 and 8 of culture ( 39 86 ; P