Non-surgical Transfer Research Articles

209 VIABILITY OF PORCINE EMBRYOS VITRIFIED BY THE METAL MESH VITRIFICATION METHOD AFTER SURGICAL OR NONSURGICAL TRANSFER

The aim of the present study was to evaluate viability of porcine embryos vitrified by the metal mesh vitrification (MMV) method after surgical or nonsurgical transfer. Prepubertal gilts were treated with eCG and hCG (= Day 0), and then inseminated artificially. Expanding blastocysts that were about 200 �m in diameter were collected on Day 7. The embryos were equilibrated in 7.5% ethylene glycol (EG) + 7.5% DMSO + 20% fetal bovine serum (FBS) in PBS at 37�C for 5 min, and then transferred into 15% EG + 15% DMSO + 0.6 M trehalose + 20% FBS in PBS for 1 min. Embryos in groups of 5 were transferred in a minimum volume of the vitrification solution (less than 1 �L) onto stainless steel mesh (75 �m screen size, 1.5 mm in width by 10 mm in length, with a 3-mm right-angle bend), and then plunged into liquid nitrogen. The mesh was stored in a 1.8-mL cryotube submerged in liquid nitrogen. Warming and dilution were performed by moving the mesh from liquid nitrogen into 0.5 M trehalose + 20% FBS in PBS at 37�C for 5 min. Embryos were rinsed twice in NCSU37 + 10% FBS (mNCSU37) for 5 min. After being vitrified, embryos in groups of 20 per recipient were suspended in modified NCSU37 medium and then transferred into gilts either by surgical transfer (5 gilts) or by nonsurgical transfer (6 sows). For surgical transfer, embryos suspended in 0.1 mL of medium were transferred into the uterine horn at 15 cm above the uterine body, which was about 35 cm from the external uterine orifice. For nonsurgical transfer, an intrauterine catheter made from polyethylene tubing (1.2 m long, 3.0 mm outer diameter, 0.5 mm inner diameter) was used. A spiral guide inserted through the vagina into the cervix was used to guide the catheter into one uterine horn. The catheter was moved through the cervix and along the uterine horn. Then, embryos suspended in 1 mL of medium were transferred. Pregnancy was assessed by ultrasonography at 30 days post-estrus. With surgical transfer, 4 of 5 recipients became pregnant, and 3 gilts farrowed a total of 21 (10, 8, 3) live piglets; the fourth gilt aborted one fetus on Day 34. With nonsurgical transfer, 3 of 6 sows became pregnant. The present study demonstrates that vitrified porcine embryos can develop after both surgical and nonsurgical transfer to recipients. As a practical measure, nonsurgical transfer of vitrified porcine blastocysts may be used instead of surgical transfer.

207 INFLUENCE OF THE INTERVAL BETWEEN THAWING TO TRANSFER ON PREGNANCY RATES OF FROZEN - THAWED DIRECT-TRANSFER EMBRYOS

An experiment was designed to evaluate the effect of the interval between thawing to deposition of the embryo into the uterine horn on pregnancy rates of in vivo-produced frozen–thawed embryos in 1.5 M ethylene glycol (direct transfer). Data were collected from 1122 embryo transfers performed in the same farm (Estancia El Mangrullo, Lavalle, Santiago del Estero, Argentina) during the spring and summer of 2004/05 and 2005/06 (6 replicates, ambient temperature between 20 and 40�C). Recipients used in all replicates were non-lactating, cycling, multiparous Bos taurus � Bos indicus crossbred cows with body condition score between 3 and 4 (1 to 5 scale) that were synchronized using fixed-time embryo transfer protocols. Briefly, the synchronization treatments consisted of the insertion of a Crestar ear implant (Intervet, Sao Paulo, Brazil) or a progesterone-releasing device (DIB; Syntex SA, Buenos Aires, Argentina), plus 2 mg of estradiol benzoate (EB; Syntex) intramuscularly (IM) on Day 0, and 400 IU of eCG (Folligon 5000; Intervet, or Novormon 5000; Syntex) IM plus 150 �g d-cloprostenol IM (Preloban; Intervet, or Ciclase; Syntex) on Day 5. Progestin devices were removed on Day 8 and all cows received 1 mg of EB IM on Day 9. All cows were examined by ultrasonography on Day 16 and those with a luteal area >76 mm2 (by calculating the area of the CL minus the area of the cavity) received, on Day 17, frozen–thawed embryos by nonsurgical transfer. All embryos were Grade 1, and all were frozen in 1.5 M ethylene glycol at the Embryo Plus Laboratory (Brits, South Africa). After being stored in liquid nitrogen, the embryos were plunged directly (no air thawing) in a 30�C water bath for 30 s, and then transferred to the recipient cows by either one of two technicians. Based on the interval between thawing and transfer, the transfers were classified as being in one of 3 groups: Group 1: <3 min; Group 2: 3 to 6 min; and Group 3: 6 to 16 min. The main reason for delayed transfers beyond 6 min was the replacement of one recipient for another because of difficulty in threading the cervix (1% of the total transfers) or a recipient falling down into the chute or with very bad disposition and behavior. Pregnancy was determined by ultrasonography 28 to 35 days after fixed-time embryo transfer, and data were analyzed by logistic regression. There were no effects of replicate, technician, CL area, recipient body condition score, embryo stage, and time from thawing to transfer on pregnancy rates. Pregnancy rates in the 3 thawing to transfer intervals were: Group 1: 215/385, 55.8%; Group 2: 372/655, 56.8%; Group 3: 42/82, 51.2%; P > 0.6. These results may be interpreted to suggest that there is no significant effect of time from thawing to transfer (up to 16 min) in direct transfer embryos using Bos taurus � Bos indicus recipients transferred at a fixed time.

215 COMPARISON BETWEEN CONVENTIONAL DIRECT TRANSFER FREEZING AND VITRIFICATION FOR THE CRYOPRESERVATION OF IN VIVO EMBRYOS FROM BRAHMAN CATTLE

There is a need to develop an efficient cryopreservation technique for Brahman cattle embryos that would lead to improved success in the propagation of this breed. The objective of this study was to compare the post-thaw pregnancy rates in recipient cows after nonsurgical transfer of Brahman in vivo-derived embryos frozen in ethylene glycol (EG) and by a new vitrification (VT) method. Prior to the initial in vivo study, hatching rates were recorded 72 h post-thaw on 3 treatment groups (No treatment: 43/55, 78%; EG: 39/83, 47%; and VT: 33/103, 32%) of IVF embryos to determine if development could be achieved. This warranted Phase I: In vivo embryos were produced by multiple ovulation and nonsurgical embryo collections from Brahman cows 7 days post-estrus and AI. Embryos in morula/blastocyst stage of development were graded and maintained in holding medium (Vigro; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) after collection, until randomly allocated for EG or VT protocols. Vitrification was performed using experimental media provided by a commercial company (V1: Vigro + 5 M EG; V2: Vigro + 7 M EG + 0.5 M Galactose + Ficoll; and Diluent: Vigro + 0.5 M Galactose) and by direct plunging into LN2 (Walker et al. 2005 Reprod. Fertil. Dev. 17, 153). Freezing was performed at 0.5�C min-1 from -6�C to -32�C, using a commercially available medium (Vigro Freeze Plus; Bioniche). All embryos were packaged in sterile 0.25-mL plastic straws to allow for direct transfer (DT). Embryos were stored in LN2, and later thawed and nonsurgically transferred to synchronized recipient cows on Day 7 of their cycle. EG straws were air-thawed 5 s and then thawed in 30�C H2O for 10 s. VT straws were air-thawed 10 s and then thawed in 37�C H2O for 20 s prior to shaking them down to mix columns. Pregnancy results evaluated by ultrasound on Day 35 for the presence of a fetus from 4 replicates were 27% for EG (3/11) and 8% for VT (1/13) embryos. Problems with VT straws cracking during air thawing led to a second trial (Phase II) which changed the direct plunging technique for VT to a vapor freeze of 1 to 15 min prior to plunging into LN2. Pregnancy results from 16 transfers were 38% for EG (3/8) and 50% for VT (4/8) embryos, with no straws cracking. Although the number of observations is small, the results obtained showed that VT-DT is satisfactory for producing pregnancies comparable to EG-DT. Further research is required to confirm the results obtained in this preliminary study, and to test whether this method will allow the successful production of pregnancies under different environmental and field conditions.

134 CONCEPTION RATES OF IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED IN 6% GLYCEROL AND TRANSFERRED BY THE DIRECT METHOD

Ethylene glycol has been used as the standard cryoprotectant for direct transfer of bovine embryos due to its high permeability. But Merton et al. reported that cryoprotectivity of glycerol for bovine embryos was superior to that of ethylene glycol (2001 Theriogenology 55, 312 abst). We previously reported that nonsurgical transfer of in vivo-derived bovine embryos cryopreserved in a lower concentration (5%) of glycerol and thawed by stepwise method resulted in a 55.4% conception rate, whereas direct transfer without removal of cryoprotectant showed only a 45.1% conception rate (Takada et al. 2005 Jpn. J. Embryo Transfer 27, 59–64). In this experiment, survival and conception rates of in vitro-produced (IVP) bovine embryos cryopreserved in 6% glycerol solution (GLY) were compared to those of embryos cryopreserved in 10% ethylene glycol plus 0.1 M sucrose solution (EG). Cumulus–oocyte complexes were matured and fertilized according to Numabe et al. (2000 Theriogenology 54, 1409–1420). Presumed zygotes were cultured in mSOF supplemented with 5% calf serum (CS) and 0.25% linoleic acid albumin at 38.5�C under 5% CO2, 5% O2, 90% N2 for 7 days. At the expanded blastocyst stage, embryos were placed in GLY or EG in PBS supplemented with 20% CS for 15 min at room temperature and loaded into 0.25-mL straws. Straws were placed directly into an alcohol freezer. When the cryoprotectant was GLY, straws were seeded at -4.0�C, held for 10 min, cooled at 0.5�C min to -30.5�C, and then plunged into liquid nitrogen. When the cryoprotectant was EG, the seeding point was -7.5�C, and the plunging point was -34.0�C, but the rest of the protocol was the same as for GLY. In Exp. 1, thawing in both groups was done in a 30�C water bath, and the contents were directly rehydrated in PBS with 20% CS. Thawed embryos were cultured in mSOF with 5% CS for 24 h to assess embryo survival rate, based on the re-expansion of the blastcoele and on their hatching ability. In Exp. 2, embryos in both groups were thawed and transferred to synchronous recipients without removing the cryoprotectant. Data were analyzed using chi-square analysis. In Exp. 1, the developmental rates of post-thaw embryos were similar in GLY (46/52, 88.5%) and EG (45/52, 86.5%); however, the hatching rate was significantly higher (P < 0.05) in embryos cryopreserved in EG (26/52, 50.0%) than in GLY (15/52, 28.8%). In Exp. 2, the conception rates of embryos were similar in both groups, GLY (7/15, 46.7%) and EG (6/15, 40.0%). In conclusion, after direct rehydration of embryos, the developmental ability of IVP bovine embryos cryopreserved in EG was superior to that of embryos cryopreserved in GLY in vitro. However, conception rates in vivo were similar in both groups. These results suggest that a lower concentration of glycerol might be still useful as a cryoprotectant for direct transfer of IVP bovine embryos.

Application Study of Intracytoplasmic Sperm Injection for Golden Hamster and Cattle Production

This paper describes several technical improvements and our results in hamster intracytoplasmic sperm injection (ICSI), hamster round spermatid injection (ROSI) and bovine ICSI. The hamster is the mammalian species in which ICSI was first tried to produce fertilized oocytes. However, until recently, no live offspring following ICSI have ever been obtained. We reported the birth of live offspring following hamster ICSI. Improved points to success were 1) performing hamster ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under microscope with a red light source and 2) injecting sperm heads without acrosomes. Under controlled illumination, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally, cleaved, and developed into morulae. Nine live offspring (19%) were born by transfer of hamster ICSI-derived embryos. Furthermore, we reported the birth of live offspring following hamster ROSI. About 70% of oocytes injected with round spermatids broken before injection were fertilized normally and about half of them developed to morulae and blastocysts. Three (5%) live young were born by transfer of hamster ROSI-derived embryos. On the other hand, in cattle, the main improvements were 1) injection of spermatozoa immobilized by scoring their tail just before injection into oocytes, and 2) additional ethanol activation 4 h after ICSI. About 70% of oocytes injected were activated 4 h after ICSI, and about 30% of them developed to blastocysts. Twenty-four live calves (39%) were born by non-surgical transfer of ICSI-derived embryos. Those results shows that, at present, live offspring are able to be obtained following hamster ICSI, ROSI and bovine ICSI, but further improvement is required due to higher production efficiency of offspring.

The Holstein cow in embryo transfer today as compared to 20 years ago

Embryo transfer practice and results were examined over a 20-year period in Holstein cows and heifers within four commercial embryo transfer programs located in different areas of North America. Mean embryo production per collection decreased ( P < 0.05) in one program over time, but not in the other three. Changes in the type of cows entering embryo transfer programs, the number of times they were superstimulated and changes in the brands of gonadotropins used for superstimulation all complicated the analysis of embryo production over time. Data reveal higher pregnancy rates ( P < 0.001) following transfer of embryos into Holstein heifers than into lactating dairy cows. It is not clear whether pregnancy rates have decreased over time as a result of the change from surgical to non-surgical embryo transfer. In the two programs in which pregnancy rates were analyzed, there was a decrease ( P < 0.001) when non-surgical transfers were adopted in one program, while no change occurred in the other. One of the biggest changes in all programs was that more than 50% of embryos recovered from donors are now frozen after collection, whereas the majority were transferred fresh 20 years ago.

The Development and Application of the Modern Reproductive Technologies to Horse Breeding

Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallion semen, and parallel improvements in ovulation induction and synchrony, exogenous gonadotrophic stimulation of multiple fertile ovulations and simplified, more efficient methods for non-surgical transfer of embryos to recipient mares, coupled with relaxation of breed society registration restrictions, have together contributed to a similar upsurge in the application of embryo transfer to all breeds and athletic types of horses worldwide, with the continuing and notable exception of the Thoroughbred. Although conventional in vitro fertilization remains something of an unjumped fence in equids, other modern breeding technologies like hysteroscopic low-dose insemination, fluorescence-activated sex sorting of stallion spermatozoa, between-species embryo transfer, embryo freezing and bisection, transvaginal ultrasound-guided oocyte collection, intracytoplasmic sperm injection for fertilization (ICSI), gamete intrafallopian transfer (GIFT) and now nuclear transfer (cloning), have all been applied to equids with encouraging success. Cloning, especially, holds enormous promise for the Sporthorse industry to re-create champion geldings in stallion form for breeding purposes.

Efectos sobre la fertilidad bovina de las técnicas de manipulación de embriones

During the past thirty years, basic and experimental studies on classical (superovulation; non-surgical recovery and transfer of cattle embryos) and advanced embryo technologies (in vitro embryo production; cloning by somatic cell nuclear transfer) have generated structural and functional information on oocyte development and quality, fertilisation and conceptus development. This information has provided new insight, not only into these technologies per se but also into the factors contributing to fertility in cattle. It is now known that the peripheral and follicular endocrine profiles have a profound influence on the subsequent developmental competence of the embryo. It is also well established that manipulation of the oocytes or embryos may adversely affect embryonic and foetal development, leading to the so-called 'large offspring syndrome'. Information from such studies has alerted scientists to the importance of epigenetics in cattle reproduction.

192 THE USE OF FIXED-TIME TECHNIQUES AND eCG TO SYNCHRONIZE RECIPIENTS for FROZEN - THAWED BOVINE IVF EMBRYOS

Two experiments were designed to investigate the use of eCG in a fixed-time embryo transfer (FTET) protocol for Chinese Yellow crossbred recipients receiving in vitro-produced (IVP) bovine embryos. In Experiment 1, cows were observed for spontaneous estrus (Group 1) or following 500 �g cloprostenol (PGF; Schering-Plough Animal Health, Montreal, Quebec, Canada; Group 2) with nonsurgical embryo transfer 7.5 or 8.0 days later. Cows in Groups 3, 4, and 5 were treated with a CIDR insert (Bioniche Animal Health, Beijing, China), 2 mg estradiol benzoate (EB), and 50 mg progesterone (Sigma-Aldrich, Canada) i.m. on Day 0, PGF on Day 5, CIDR removal on Day 7, and 1 mg EB i.m. 24 h later with nonsurgical transfer of Holstein IVP embryos 8.5 days later, without estrus detection. In addition, cows in Group 3 received no further treatment, whereas cows in Group 4 received 400 IU eCG i.m. (Pregnecol; Bioniche) on Day 5, and cows in Group 5 received eCG on Day 7. Holstein embryos were produced utilizing slaughterhouse ovaries and standard IVF procedures. Expanded blastocysts of quality grade 1 (IETS) were cryopreserved in 10% ethylene glycol and 20% fetal bovine serum using standard procedures. Straws were thawed in a 30�C water bath, and embryos were expelled directly into holding medium and evaluated prior to nonsurgical transfer (NT) ipsilateral to the corpus luteum (CL). Pregnancy was diagnosed ultrasonically 30 d later. Although pregnancy rates, based on numbers of recipients synchronized, numerically favored the administration of eCG on Day 5, there were no significant differences (P = 0.40) among groups (Group 1: 15.4%, n = 52; Group 2: 20.0%, n = 50; Group 3: 19.2%, n = 99; Group 4: 28.1%, n = 96; Group 5: 21.3%, n = 75). In Experiment 2, Chinese Yellow crossbred cattle were synchronized with a CIDR insert plus estradiol and progesterone on Day 0, PGF on Day 5, CIDR removal on Day 7 and estradiol on Day 8, and were randomly assigned to received no further treatment (Group 1; n = 400) or an injection of 400 IU eCG on Day 5 (Group 2; n = 391). Recipients with a detectable CL received a frozen-thawed IVP Holstein embryo by NT 8.5 days after the second injection of estradiol without estrus detection, as in the first experiment. Pregnancy diagnosis was done ultrasonically 30 days later. Although CL size, cow age, and embryo quality, prior to transfer, were recorded, no effects on pregnancy rates were demonstrated (P = 0.30). Pregnancy rates, based on recipients receiving embryos, did not differ (P = 0.5) between groups (Group 1: 21.4%, n = 154; Group 2: 24.5%, n = 290). Overall pregnancy rates (based on the total number of recipients synchronized) were significantly higher (P &lt; 0.001) in Group 2 (eCG; 18.2%) than in Group 1 (no eCG; 8.3%), because of the significantly higher (P &lt; 0.03) percentage of recipients used following treatment with eCG (74.2% vs. 38.5%). Results indicate that the administration of eCG on Day 5 of an 8-d synchronization protocol for FTET of frozen-thawed bovine IVP embryos will improve pregnancy rates in beef recipients, especially those of marginal quality.

39 CHROMOSOME ABNORMALITIES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY 'HANDMADE CLONING'

Embryos produced by nuclear transfer using somatic cells as donors exhibit a lower developmental competence compared to in vivo developed and IVM/IVF/IVC embryos. The so-called handmade somatic cell nuclear transfer (HSCNT or handmade cloning) that was recently presented (Vajta et al. 2003 Biol. Reprod. 68, 571–578) has, however, improved the reconstructed embryo and subsequent blastocyst rate. Further, the high total cell number (n = 216) and the high ratio of cells allocated to the inner cell mass (35%) as well as the initial pregnancy rate of 48% on Day 28 following nonsurgical transfer of HSCNT embryos indicated a high average quality. But still the pregnancy loss of HSCNT embryos was high and resulted in a birth rate of 8%. The aim of this study was to estimate the chromosomal variation in HSCNT embryos using fluorescent in situ hybridization (FISH) and to evaluate this as an additional parameter of embryo quality. Nuclei from 49 Day 7 HSCNT embryos from five independent trials were isolated by hypotonic treatment and fixed. Then the nuclei were hybridized with differentially labelled cJAB8 and p33E39 probes that hybridize specifically to the centromeric region of chromosome 6 and 7 (Viuff et al. 2000 Biol. Reprod. 63, 1143–1148). A total of 6715 nuclei were analyzed for chromosomal abnormalities, and the percentage of nuclei with false negative scores represented 1.8%. Only 4.1% of the embryos (2 of 49) had a completely normal diploid composition, while the remaining 47 embryos exhibited different types of mixoploidy. Of the 47 mixoploid embryos, 87% contained more than one type of chromosomal variation with diploid/triploid/tetraploid being the most frequent constitution (43% of the 47 mixoploid embryos). No pure polyploid embryos were observed. The percentages of HSCNT embryos in the groups of 0%, 1–25%, and 26–100% polyploid cells were 4.1%, 67.5%, and 28.6%, respectively. This is significantly higher than the corresponding figures produced and analyzed by comparable methodology of bovine IVM/IVF/IVC embryos (Viuff et al. 1999 Biol. Reprod. 60, 1273–1278). However, 71.4% of the HSCNT embryos contained less than 25% polyploid cells; this low level may not have compromised the developmental competence of these embryos.

Perspectives for artificial insemination and genomics to improve global swine populations

Civilizations throughout the world continue to depend on pig meat as an important food source. Approximately 40% of the red meat consumed annually worldwide (94 million metric tons) is pig meat. Pig numbers (940 million) and consumption have increased consistent with the increasing world population (FAO 2002). In the past 50 years, research guided genetic selection and nutrition programs have had a major impact on improving carcass composition and efficiency of production in swine. The use of artificial insemination (AI) in Europe has also had a major impact on pig improvement in the past 35 years and more recently in the USA. Several scientific advances in gamete physiology and/or manipulation have been successfully utilized while others are just beginning to be applied at the production level. Semen extenders that permit the use of fresh semen for more than 5 days post-collection are largely responsible for the success of AI in pigs worldwide. Transfer of the best genetics has been enabled by use of AI with fresh semen, and to some extent, by use of AI with frozen semen over the past 25 years. Sexed semen, now a reality, has the potential for increasing the rate of genetic progress in AI programs when used in conjunction with newly developed low sperm number insemination technology. Embryo cryopreservation provides opportunities for international transport of maternal germplasm worldwide; non-surgical transfer of viable embryos in practice is nearing reality. While production of transgenic animals has been successful, the low level of efficiency in producing these animals and lack of information on multigene interactions limit the use of the technology in applied production systems. Technologies based on research in functional genomics, proteomics and cloning have significant potential, but considerable research effort will be required before they can be utilized for AI in pig production. In the past 15 years, there has been a coordinated worldwide scientific effort to develop the genetic linkage map of the pig with the goal of identifying pigs with genetic alleles that result in improved growth rate, carcass quality, and reproductive performance. Molecular genetic tests have been developed to select pigs with improved traits such as removal of the porcine stress (RYR1) syndrome, and selection for specific estrogen receptor (ESR) alleles. Less progress has been made in developing routine tests related to diseases. Major research in genomics is being pursued to improve the efficiency of selection for healthier pigs with disease resistance properties. The sequencing of the genome of the pig to identify new genes and unique regulatory elements holds great promise to provide new information that can be used in pig production. AI, in vitro embryo production and embryo transfer will be the preferred means of implementing these new technologies to enhance efficiency of pig production in the future.

Comparison of four synchronization protocols for fixed-time bovine embryo transfer in Bos indicus × Bos taurus recipients

The objective was to evaluate the effects of 400 IU of eCG given on Days 5 or 8 of an estrus synchronization protocol with progesterone-releasing intravaginal devices (PRID) and estradiol benzoate (EB), in recipients for fixed-time embryo transfer. A secondary objective was to determine the effects of injectable progesterone (given concurrent with EB treatment). Three-hundred-and-four crossbred Bos taurus × Bos indicus beef heifers were randomly assigned to one of four treatment groups (2 × 2 factorial design). At unknown stages of the estrous cycle (Day 0), all heifers received a progesterone-releasing intravaginal device (PRID), plus 2 mg of EB i.m., with or without a concurrent treatment of 50 mg of progesterone i.m. Heifers were further subdivided to receive 0.15 mg of d-cloprostenol (PGF) i.m. and 400 IU of eCG i.m. on Days 5 or 8. In all heifers, intravaginal devices were removed on Day 8 and 1 mg of EB was given i.m. on Day 9 (Day 10 was arbitrarily considered the day of estrus). On Day 17, all heifers with >1 CL or a single CL with a diameter ≥18 mm (based on ultrasonographic examination), received an in vitro produced (IVP) embryo by non-surgical transfer. On Day 17, there was an effect of day of eCG administration on the number of CL (1.35±0.08 versus 1.13±0.04, for Day 5 versus Day 8, respectively; P=0.02) and (in a subset of 154 heifers) mean (±S.E.M.) plasma progesterone concentrations (2.41±0.26 versus 1.74±0.19 ng/mL; P=0.03). Although the proportion of recipients transferred/treated and pregnant/transferred did not differ among groups, the proportion of recipients pregnant/treated tended ( P=0.1) to be higher in heifers treated with eCG on Day 5 versus Day 8 (47.0% versus 40.7%, respectively). Progesterone treatment had no significant effect. In conclusion, treatment with eCG (and d-cloprostenol) on Day 5 significantly increased the number of CL and plasma progesterone concentrations and tended to increase pregnancy rates, although progesterone treatment had no significant effect.

Quality of porcine blastocysts produced in vitro in the presence or absence of GH.

GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced blastocysts have lower cell numbers than in vivo blastocysts and exhibit higher incidences of apoptosis. Therefore we investigated the effects of 100 ng GH/ml NCSU23 medium during in vitro culture of presumptive in vitro fertilized sow zygotes on embryo development and blastocyst quality (defined by diameter, cell number, apoptosis and survival after non-surgical transfer). In vivo produced blastocysts were analysed concurrently as a reference value. GHR was expressed in embryos from the 2-cell to blastocyst stages. GH had no effect on blastocyst development or cell numbers, but increased the mean blastocyst diameter. The incidence of apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), was decreased by GH, but when non-TUNEL-labelled apoptotic fragmented nuclei were included, no difference was seen. GH appeared to slow down the progression of apoptosis though. In vivo produced blastocysts presented no apoptotic nuclei, and contained higher cell numbers and larger diameters. Pregnancy rates on day 11 were similar for all groups, but survival was poorer for in vitro than in vivo produced blastocysts. In this study GH appeared to be beneficial only from the blastocyst stage, but the presence of GHR from early cleavage stages nevertheless indicates a role for GH throughout porcine embryo development and deserves further investigation.

169 EMBRYO TRANSFER OF SEXED/VITRIFIED IVF EMBRYOS IN CATTLE: PREGNANCY COMPARISON AFTER SINGLE AND DOUBLE TRANSFERS

Advancement in sperm sorting technology combined with vitrification of in vitro produced bovine embryos will promote cattle breeding and production. The objective of this study was to evaluate pregnancy and embryo loss after embryo transfer (ET) of sexed/vitrified embryos with one bilaterally (double transfer, 2 embryos) v. ipsilaterally (single transfer, 1 embryo) into the recipient. Bovine oocytes collected from slaughterhouse ovaries or ovum pickup were matured for 20-22 h, then subjected to IVF using Brackett and Oliphant BO procedures with sorted X-sperm, and cultured with our standard culture system. Expanded blastocysts with tight compaction of the inner cell mass (quality 1) were selected on Day 7 for cryopreservation via liquid nitrogen surface vitrification (LNSV; Xu et al. 2006 J. Dairy Sci. 89, 2510-2518). Embryo transfer was performed for 3 replicates in Navasota, Texas, in April 2009. Prior to ET, embryos were warmed and subsequently washed several times in warming, dehydration solution and base medium. Some of sexed/vitrified embryos were cultured for 3 days post-warming to determine the survivability. The treatments were as follows: (1) vitrified-single transfers, 1 embryo was transferred into the horn ipsilateral to CL; (2) vitrified-double transfers, 1 embryo was transferred into each uterine horn by nonsurgical transfer; and (3) fresh-single, 1 fresh embryo was transferred into the horn ipsilateral to CL (control) to a synchronous recipient on Day 7. Pregnancy was determined by ultrasound monitoring on Day 35, and palpation per rectum on Day 75 after transfer. The pregnancy data were analyzed by General Linear Model analysis (SPSS 11.0, SPSS Inc., Chicago, IL, USA). The survival rate of vitrified IVF embryos reached to as high as 97.6% (n = 42) 2 h post-warming, and hatching rate was 85.7% after 3 days culture in vitro. The data (Table 1) showed that there was no difference in Day 35 pregnancy rate among vitrified-double, vitrified-single, and fresh ET control groups. However, on Day 75 post-ET, there was a significantly higher fetal loss found in the vitrified-double transfer group (41.1%) compared to those of vitrified-single transfers (16.6%) and fresh-single group (11.9%) (P &lt; 0.05). The pregnancy rate on Day 75 of 51.4% achieved with vitrified-single transfers was comparable to the 43.3% achieved with the fresh-single control transfers but was significantly higher than the 31.1% of the vitrified-double transfer group. This study demonstrated that double embryo transfers can aggravate high fetal loss and/or abortion when sexed IVF embryos are transferred, and ET with 1 sexed/vitrified embryo per recipient is sufficient to establish satisfactory pregnancy, comparable to that achieved with fresh embryos. Table 1.Pregnancy and fetal loss of sexed/vitrified bovine IVF embryos following single and double transfers Supported by USDA/CSREES-SBIR: 2006-03069 Phase II to F. Du.

Birth of piglets through the non-surgical transfer of blastocysts produced in vitro.

In this study, we attempted to produce piglets by non-surgically transferring blastocysts produced in vitro, using a flexible catheter as the transfer instrument. Cumulus-oocyte complexes (COCs) were aspirated from the follicles of ovaries obtained at a local slaughterhouse. They were then matured in modified North Carolina State University (NCSU)-37 medium for 44-46 h and fertilized in porcine gamete medium (PGM). Ten hours after in vitro fertilization (IVF), presumptive zygotes were removed from the cumulus cells and cultured in porcine zygote medium (PZM)-5. Blastocysts were cultured for five days after IVF and, using a catheter for deep intrauterine insemination without sedation, they were transcervically transferred into the uterine horn of six recipients (45-50 blastocysts/recipients) whose estrous cycles were synchronized, at 5 days after human chorionic gonadotropin (hCG) injection. Of the six recipients, one sow became pregnant and farrowed seven piglets (four live piglets) 119 days after hCG injection. The body weight at birth of the newborns ranged from 0.8 to 1.4 kg. These results indicate that it is possible to obtain piglets by transcervically transferring blastocysts produced by IVF and in vitro cultures in chemically defined media.

74PREGNANCY ESTABLISHMENT OF NUCLEAR TRANSFERRED EMBRYOS AFTER VITRIFICATION AND EMBRYO TRANSFER IN CATTLE

The success of cryopreservation of bovine cloned embryos is important to commercial application of somatic nuclear transfer (NT) in the dairy and beef industry, especially for solving the problem of embryo transfer (ET) between fresh NT embryos and recipients. This experiment was conducted to test the possibility of establishing pregnancy by embryo transfer of vitrified bovine NT embryos. Oocytes were aspirated from antral follicles of slaughterhouse ovaries, and subsequently cultured in maturation medium for 20h. Cumulus cells were then denuded from the oocytes by vigorous vortexing for later enucleation and donor nuclear transfer. Skin fibroblast cells used for NT were derived from cultured ear explants taken from an elite dairy cow, and cumulus cells were cultured from the cumulus-oocytes complexes collected by ultrasound-guided transvaginal retrieval. Fibroblasts and cumulus cells were cultured at passage 5 or 6 in 10% FBS DMEM at 37°C in 5% CO2 humidified air, and used as nuclear donors. After donor cell transfer, somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0kVcm−1 for a duration of 10μs/pulse. Fused embryos were activated in 10mgmL−1 cycloheximide in M199+7.5% FBS for 4h. Embryos were cultured in CR1aa plus 3mgmL−1 BSA for 2 days (initiation of activation=Day 0) at 39°C, 5% CO2, 5% O2 and 90% N2, and then cultured on bovine cumulus monolayers in CR1aa medium supplemented with 7.5% FBS for 5 successive days. Expanding and hatching blastocysts on Day 7 were selected for cyropreservation via liquid nitrogen surface vitrification (LNSV). Vitrification solution contained HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol and dimethylsulphoxide. A droplet of 1–2μL vitrification solution containing two blastocysts was directly dropped onto a cooled surface within 30s after 3min incubation in equilibration solution. Vitrified NT embryos were kept at −150°C vapor phase until recipients were synchronized to Day 7 for ET. Embryos are warmed and subsequently washed several times in rehydration solutions and M199+7.5% FBS medium. The warmed embryos from initial trials were cultured for 2h to evaluate their viability after cryopreservation. Non-surgical transfer was carried out to transfer two embryos to a synchronous recipient, and pregnancy was determined by palpation via rectum around Day 70 of transfer. After warming of vitrified embryos, similar high survival rates were achieved in NT embryos derived from either cumulus (93.8%, n=16), or fibroblast cells (95.8%, n=48) as nuclear donors, respectively (P&amp;gt;0.05). The pregnancy data (Table 1) on Day 70 of gestation indicated that there were no significant differences among ET groups with fresh NT blastocysts, vitrified cumulus NT and fibroblast NT embryos (P&amp;gt;0.05). This study demonstrates that cryopreserved bovine NT embryos via LNSV vitrification can maintain an in vivo developmental viability comparable to freshly produced NT counterparts. Further developmental potential of vitrified NT embryos to term is under investigation. Table 1 Pregnancy outcomes of vitrifield bovine NT embryos after embryo transfer

183PREGNANCY RATE FOLLOWING TRANSFER OF IN VITRO- AND IN VIVO-PRODUCED BOVINE EMBRYOS TO LH-TREATED RECIPIENTS

The objective was to evaluate the effect of pLH treatment on pregnancy rates in recipients receiving in vivo- or in vitro-produced bovine embryos. Heifers (n=37) and lactating (n=28) and non-lactating (n=150) beef cows were treated at random stages of the cycle with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day −9, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day —2 and GnRH on Day 0 (66h post-PGF; without estrus detection). Cattle were placed at random, by class, into three groups: no further treatment (Control; n=71), or 12.5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) on Day 5 (n=72) or on Day 7 (n=72) after the second GnRH. On Day 7, cattle with a CL &amp;gt;10mm in diameter (determined ultrasonically) received in vivo-produced, fresh (Simmental) or frozen (Holstein), or in vitro-produced frozen (Holstein) embryos (embryo type balanced among groups). Embryos were cryopreserved in 10% ethylene glycol; in vivo-produced frozen embryos were thawed 5 to 10s in air, 15s in a water-bath at 30°C and then “direct-transferred” nonsurgically. In vitro-produced frozen embryos (donated by IND Lifetech Inc., Delta, British Columbia, Canada) were thawed in a water-bath at 27°C for 10s and placed in ViGro Holding Plus medium (AB Technology, Pullman, WA, USA) at room temperature, evaluated and then transferred nonsurgically. Pregnancy was determined by ultrasonography on Day 35. Data were analyzed with CATMOD, chi-square and GLM procedures (SAS Institute, Cary, NC, USA.). Twenty cattle (9.3%) did not receive embryos; five heifers had cervical problems, and five heifers and 10 cows did not have a CL &amp;gt;10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P&amp;gt;0.05) of treatment, embryo type (or interaction) or class of recipient on pregnancy rate (overall, 44.1%, 86/195; Table 1). Similarly, mean (±SD) CL diameter and luteal area did not differ (P&amp;gt;0.05) among groups or between pregnant and open recipients (overall, 22.0±3.4mm and 352.0±108.7mm, respectively). However, recipients with a CL diameter ≥18mm tended (P&amp;lt;0.1) to have a higher pregnancy rate (45.8 vs 25.0%). In a subset of 40 recipients examined ultrasonically on Day 12, 50% of those treated on Day 5 and 70% of those treated with pLH on Day 7 had two CL. In summary, overall pregnancy rate in GnRH-synchronized recipients receiving in vitro- or in vivo-produced embryos by nonsurgical transfer was 44.1%. Embryo survival to Day 35 was not affected by type of embryo or treatment with pLH 5 or 7 days after ovulation. Table 1 Pregnancy rate in recipients on Day 35 based on pLH treatment and embryo-type

180EFFECT OF TIME OF ECG TREATMENT ON PREGNANCY RATES IN BOS INDICUS×BOS TAURUS RECIPIENTS SYNCHRONIZED WITH PROGESTERONE VAGINAL DEVICES AND TRANSFERRED WITHOUT ESTRUS DETECTION

It has been shown recently that treatments with progesterone (P4)-releasing devices combined with estradiol benzoate (EB) plus P4 on Day 0, eCG and PGF on Day 5 and a second application of EB one day after device removal (Day 9) can be used successfully to transfer bovine embryos at a self-appointed time, without the necessity of estrus detection. Although the treatment solved one of the major problems in recipient management, estrus detection, it requires handling the recipients at least five times for treatments and embryo transfer. An experiment was designed to evaluate whether reducing one day of handling, by the administration of eCG and PGF at the time of removal of the P4 device (Day 8), results in comparable pregnancy rates than giving eCG on Day 5. A secondary objective was to determine the effect of injectable P4 at the time of device insertion plus EB treatment. Crossbred Bos taurus×Bos indicus beef heifers (n=301) were randomly assigned to 4 treatment groups in a 2 by 2 factorial design. All Heifers received a P4 device (DIB, Syntex, Argentina) plus 2mg EB i.m. (Syntex) at unknown stages of the estrous cycle (Day 0), with or without 50mg of P4 given i.m. at the same time. Heifers were further subdivided to receive PGF (0.150mg d-cloprostenol, Prolise, Tecnopec, Sao Paulo, Brazil) and 400IU of eCG (Novormon, Syntex) i.m. on Days 5 or 8. In all heifers, DIB devices were removed on Day 8 and 1 mg EB was administered i.m. on Day 9. Day 10 was arbitrarily considered as the day of estrus. On Day 17, heifers were bled for plasma P4 concentrations and examined by ultrasonography to determine the number of CL and their diameter. Heifers that had &amp;gt;1 CL or a single CL with diameter ≥18mm received an in vitro-produced (IVP) embryo by nonsurgical transfer performed by the same veterinarian. Pregnancy rates were determined by ultrasonography 30 days later. The effects of Day of eCG administration (Day 5 or Day 8), P4 of treatment (E2 or E2+P4) and the day-by-P4 treatment interaction on the numbers of CL and plasma P4 were analyzed by ANOVA, and the proportion of recipients selected and pregnant were analyzed using non-parametric tests (NPAR1WAY, SAS). There was no significant effect of P4 treatment or the P4-by-day of eCG interaction in any of the parameters evaluated. However, there was a significant effect of day of eCG administration on plasma P4 concentrations (Day 5=2.4±0.3 v. Day 8=1.7±0.2; P=0.03) and the number of CL (Day 5=1.4±0.1 v. Day 8=1.1±0.0; P=0.02) on Day 17. Furthermore, the proportion of recipients pregnant/treated tended (P=0.1) to be higher in heifers in the Day 5 Group (71/151, 47.0%) than in those in the Day 8 Group (61/150, 40.7%). Although delaying the eCG and PGF administration from Day 5 to Day 8 saves one trip through the chute for treatments, it resulted in lower plasma P4 concentrations and tended to decrease pregnancy rates in bovine embryo recipients synchronized with DIB devices and EB and transferred at a fixed time. Furthermore, the administration of injectable P4 at the time of DIB insertion did not affect pregnancy rates.

Fetometry and fetal heart rates between Day 35 and 108 in bovine pregnancies resulting from transfer of either MOET, IVP-co-culture or IVP-SOF embryos

The Large Offspring Syndrome has frequently been reported for in vitro produced calves. The objective of this study was to determine whether any differences in body dimensions (biparietal diameter of the cranium (BPD), cross-section of the abdomen at the insertion of the umbilical cord (CAU)) and heart rate (FHR) can be detected during the first 108 days of gestation between bovine foetuses derived from different methods of embryo production. Three groups of pregnancies with calvings at term resulted from non-surgical transfers of three types of embryos: recipients carrying an embryo obtained by standard MOET procedures ( n=25); recipients carrying an embryo produced in vitro from OPU-derived oocytes, using co-culture-medium ( n=14) or SOF-medium ( n=22). Transrectal ultrasonographic examinations were performed weekly. Ultrasound images were recorded and during off-line analysis FHR, BPD and CAU were determined. For each foetus a curve was fitted and the estimates on fixed time intervals were used as dependent variables in an analysis of variance to detect differences between the three pregnancy groups. Neither gestation length nor birth weight differed significantly between the three pregnancy groups, nor could any differences with respect to BPD, CAU or FHR be detected between Days 35 and 108 of gestation. It is concluded that no differences exist between the early development of bovine foetuses, derived from MOET, IVP-co-culture or IVP-SOF embryos, and resulting in calves with normal birth weights.

Successful non-surgical transfer of horse embryos to mule recipients.

Mules, hybrids resulting from the mating of a horse mare (Equus caballus, 2n = 64) to a Jack donkey (E. asinus, 2n = 62), are generally infertile. Five horse embryos were transferred non-surgically to two cyclic and one acyclic recipient mules. In the mares and cycling mules, oestrus and ovulation were induced with, respectively, d-cloprostenol and human chorionic gonadotrophin (hCG). The acyclic mule, on the other hand, received oestradiol benzoate when the embryo donor was showing oestrus and progesterone after the donor had ovulated and until pregnancy diagnosis. Non-surgical embryo collections were attempted on day 7 after ovulation and recovered embryos were transferred transcervically into the mules' uteri. Mules that became pregnant were blood sampled serially for equine chorion gonadotrophin (eCG), progestagen and total conjugated oestrogen concentrations until around 6 months of gestation. The three embryos transferred to the acyclic mule did not produce any pregnancies whereas both embryos transferred to the cycling mules resulted in the birth of live foals. The peak concentration and duration of secretion of eCG differed markedly between the two pregnant mules, although both animals appeared to develop secondary corpora lutea beyond day 40 of gestation, as in normal intraspecies horse pregnancy. Moreover, the rise in serum oestrogen concentrations from around day 90 was also similar to that seen in normal pregnant mares. Parturition occurred spontaneously on day 348 of gestation in both mules and the resulting colt foals developed normally to weaning. Thus, cycling mules can carry a horse conceptus after non-surgical embryo transfer and give birth to a normal mature foal.