Donor Cows Research Articles

138 CELL INTEGRITY OF HOLSTEIN AND JERSEY EMBRYOS: COMPARISON OF TWO FREEZING METHODS

The aim of this study was to compare cell integrity of Holstein and Jersey embryos using the standard method of freezing and vitrification. Embryos were harvested and processed following standard procedures approved by IETS (International Embryo Transfer Society), and then underwent two methods of freezing. (1) Standard method: embryos were put into 1.5 m ethylene glycol (EG) for 5 min at room temperature, and then at –6.5�C for 10 min for stabilization. After seeding, the temperature was lowered by 0.5�C until it reached –32�C, and then straws containing embryos were plunged into liquid nitrogen. (2) Vitrification: embryos were put for 3 min into vitrification solutions V1: 5 m EG in EmCare (ICPbio, Ltd., Auckland, New Zealand) and V2: 7 m EG, 0.5 m galactose (Sigma Chemical Co., St. Louis, MO, USA), and 18% Ficoll70 (Sigma) in EmCare for 45 s. Embryos were loaded into straws and then plunged into liquid nitrogen. De-freezing was done as follows: 5 s at room temperature, and then 30 s in water at 25�C. Vitrified embryos were de-frozen for 10 s at room temperature followed by 30 s in water at 25�C. All embryos were subjected to a combined staining, Hoechst 33342 and propidium iodide (Sigma-Aldrich Canada, Toronto, Ontario, Canada). The combined staining with these dyes makes analysis of cell death possible. Propidium iodide is specific to dead cells, whereas Hoechst stains all of the cells. Embryos were put in a PBS solution containing 20 µg mL–1 of Hoechst 33342 and propidium iodide for 15 min at 37�C and placed between a slide and coverslip. Fluorescence microscopy was then used to assess blue nuclei (live cells) and red nuclei (dead cells) of the cow embryos. For this experiment, a total of 51 grade 1 embryos (IETS classification) at early blastocyst or blastocyst stages were used (25 Holstein embryos from 4 donor cows and 26 Jersey embryos from 3 donor cows). The standard method of freezing was performed on 26 embryos (13 from Holstein cows and 13 from Jersey cows) and vitrification was performed on 25 embryos (12 from Holstein cows and 13 from Jersey cows). Embryos from a given cow (Holstein or Jersey) were evenly allocated to the standard and vitrification methods. The GLM procedure of SAS (SAS statistical software version 8; SAS Institute, Inc., Cary, NC, USA) was used to compare numbers of dead and live cells between vitrification and the standard method of freezing in Holstein and Jersey embryos. The mean numbers (�SE) of live cells of Holstein embryos were, respectively, 87.92 � 8.48 and 85.5 � 6.46 for standard method and vitrification. For Jersey embryos, means (�SE) of live cells were 94.42 � 2.6 and 73.93 � 7.39, respectively, for the standard method and vitrification. Means (�SE) of dead cells of Holstein embryos were, respectively, 5.58 � 1.37 and 13.67 � 2.91 for the standard method and vitrification. For Jersey embryos, means (�SE) of dead cells were 8.08 � 1.01 and 27.6 � 7.06, respectively, for the standard method and vitrification. In conclusion, vitrification significantly increased (P ≤ 0.05) the number of dead cells of embryos over the standard method of freezing. This effect was more evident (P ≤ 0.05) in Jersey than in Holstein embryos. The authors thank Dr. Vincent Girard for his help with statistics.

196 COMPARING OOCYTE LIPID CONTENT WITH CIRCULATING CHOLESTEROL AND TRIGLYCERIDE LEVELS OF BOS TAURUS AND BOS INDICUS DONOR COWS

Intracellular lipid content has been found to be higher in Bos indicus oocytes compared with Bos taurus oocytes (Ballard et al. 2007 Reprod. Fertil. Dev. 19, 170). The objective of this study was to further determine whether there was a correlation between oocyte lipid content and circulating cholesterol (CHL) and triglyceride (TRG) levels of mature purebred Brahman (BR; n = 11) and English breed (EN; n = 13) beef cows. All donor cows were maintained on the same ration 30 days prior to and during the oocyte collection interval. Oocytes were collected from donors at 30-day intervals (3 replicates/female) by transvaginal ultrasound-guided aspirations. Blood samples were collected to evaluate total circulating CHL and TRG levels just prior to CIDR insertion and again prior to oocyte collection. Follicle-stimulating hormone (Folltropin-V; Bioniche, Belleville, Ontario, Canada) was given twice daily for 3 days in decreasing doses beginning on Day 4 after CIDR insertion, with oocyte recovery on Day 8. The mean number of follicles aspirated/donor and oocytes recovered/donor were 12 and 11 for the BR and 18 and 7 for the EN donors, respectively. Oocytes were matured in TCM-199 with 10% fetal bovine serum + bovine LH and bovine FSH (0.01 U mL–1). After 20 h, mature oocytes with a visible polar body (BR = 242/391 or 61%, and EN = 170/258 or 65%) were denuded by vortexing in Tyrodes HEPES medium + BSA (0.03 mg mL–1). Visual color scores (1 = light, 2 = medium, 3 = dark) were then assigned to the oocytes. Buoyancy of mature oocytes was then measured by using a sucrose step-density gradient column prepared with sucrose and Dulbecco's PBS. Results from the sucrose gradients ranged from 23% (high lipids) to 35% (lower lipids). The oocytes were stained with Nile Red and evaluated by fluorescent microscopy. Oocyte images were evaluated by using Scion Image software to calculate the Nile Red units (NRU; higher NRU = higher lipid content). Serum was analyzed (Texas Veterinary Medical Diagnostic Laboratory) for CHL and TRG concentrations. Data were analyzed by one-way ANOVA. There was no difference between visual color scores for oocytes from BR and EN donors. However, BR oocytes had significantly (P < 0.05) lower buoyant density and significantly higher mean NRU scores (32% and 173) compared with oocytes harvested from EN donors (33% and 152), respectively. Serum CHL and TRG levels were both significantly higher (P < 0.05) in the BR donors (150 and 33.2) compared with the EN donors (110 and 25.5), respectively. These results suggest that serum cholesterol and triglyceride levels may be predictive of donor oocyte lipid content.

384 EFFECTS OF CONDITION SCORE ON OVARIAN RESPONSE AND TRANSFERABLE EMBRYOS IN SUPERSTIMULATED COWS IN ARGENTINA

The success of an embryo transfer program is influenced by ovarian response, number of ovulations, fertilization, and embryo viability (Armstrong 1993). It has been observed that over-conditioned, overweight donor females tend to produce fewer viable embryos per collection (Stroud and Hasler 2006 Theriogenology 65, 65–76). The condition score is one of the many intrinsic factors that affects cow reproduction. A retrospective analysis was done to compare the ovarian response, number of ovulations, fertilization, and total transferable embryos recovered of cows with different condition scores in Argentina. This research was carried out using different breed donors (n = 640; 82% Angus; 18% other) during the last 4 years in Buenos Aires province, Argentina. Donors at random stages of follicular wave with CL received an intravaginal progesterone device (DIB; Syntex, Buenos Aires, Argentina), 2 mg of estradiol benzoate, and 50 mg of progesterone (Syntex, Buenos Aires, Argentina) IM on the same day. On Day 4 after DIB insertion, superestimulatory treatment was initiated on a decreasing dose regimen of FSH (Pluset®; Callier, São Paulo, Brazil, or Folltropin®; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) as IM injections every 12 h during 4 days. On Day 6, DIBs were removed, and cows received a double dose of 2 mL of cloprostenol within 12 h. At heat detection, all donors received a dose of 2 mL of GnRH (Dalmarelin; Von Franken, Buenos Aires, Argentina) by IM injection and were inseminated 12 and 24 h thereafter. Seven days later, embryo collection was performed and ovarian response was evaluated as number of CL + anovulatory follicles by transrectal ultrasound using a 7.5-MHz transducer (Pie Medical, Maastricht, The Netherlands). The condition score was determined at this time using a 1 to 5 scale. Embryos recovered were evaluated and classified according to the IETS manual. Kruskal–Wallis test was used to compare variables among score groups, and results are shown in Table 1. Ovarian response as the number of CL and anovulatory follicles was significantly different (P = 0.0001) among groups. There is a negative correlation (−0.2194) between condition score and ovarian response. At higher condition scores, ovarian response decreased. There is a significant difference (P = 0.0001) for the number of ovulations and total number of fertilized ova among groups. Finally, the total number of transferable embryos was no different for either score; however, there is a trend toward decrease when the condition score is higher. In conclusion, these results indicate that using donors with condition scores of 2 to 3 improves the results of a superstimulatory treatment. Controlling donors' condition score by nutrition management gives us the possibility to produce more embryos per donor treatment. Table 1. Condition score, ovarian response, fertilization, and transferable embryos in donor cows in Argentina

39 BOVINE SOMATIC CELL NUCLEAR TRANSFER USING CUMULUS - OOCYTE COMPLEXES COLLECTED FROM THE IDENTICAL INDIVIDUAL BY OVUM PICKUP

We previously produced a cloned calf by nuclear transfer (NT) using cumulus cells removed from cumulus–oocyte complexes (COCs) after IVM. If both cumulus cells and oocytes are obtained identically and individually, and can be used simultaneously for NT, the production of cloned cows will be more expedient. And the cloned offspring produced from them will not exhibit the heteroplasmic mixed mtDNAs of donor cells and recipient oocytes. In this study, we examined the developmental potential of NT embryos using cumulus–oocyte complexes (COCs) collected from cows individually by ovum pickup (OPU). The cumulus cells were removed from COCs after IVM. The cumulus cells and cumulus-free MII oocytes derived from the same cow were used as donor nuclei and recipient oocytes, respectively. NT was performed as previously described (Akagi et al. 2003 Clon Stem Cells 5, 101–108). In Experiment 1, we examined the in vitro development of NT embryos using COCs collected by OPU. The aspiration of the follicles was performed once a week consecutively for 6 weeks in 6 cows (Cows A, B, C, D, E, and F) without hormone stimulation. In Experiment 2, we examined fetal development after the transfer of NT embryos. A Japanese black cow (Cow G) was used for OPU. On Day 7, 13 NT blastocysts were transferred to 7 recipient cows. The mtDNA genotypes of the donor cow and the cloned calf were analyzed by PCR-mediated single-strand conformation polymorphism analysis as previously described (Takeda et al. 2003 Mol. Reprod. Dev. 64, 429–437). The results of Experiment 1 are summarized in Table 1. Fusion rates did not differ among individual cows. However, the developmental rates of NT embryos at the blastocyst stage varied widely among individual cows, with a range of 19 to 64%. In Experiment 2, 2 of 7 recipient cows became pregnant on Day 30. One pregnant cow aborted on Day 60, and another cow calved a healthy calf. The mtDNA genotype of the cloned calf was confirmed to be identical with that of the donor cow. These results indicate that COCs from an identical individual can be used as donor nuclei and recipient oocytes for NT in order to produce female clones with the same mtDNA as that of the donor cow. Table 1.In vitro development of NT embryos using COCs collected by OPU

Effects of Bovine Somatotropin (bST) Administration Combined with Controlled Internal Drug Release (CIDR) on Embryo Quality and Pregnancy of Hanwoo (Korean Native Beef Cattle) during Commercial Embryo Transfer Program

Effects of recombinant bovine somatotropin (bST) on plasma hormonal concentration, embryo quality, and pregnancy rate were examined during the superovulation and synchronization treatment in donor and recipient cows. Hanwoos (Korean native beef cattle) were treated with controlled internal drug release (CIDR) combined with bST (CIDR+bST) or without bST (CIDR) as donor cows. The embryos recovered from donors were transferred into Holstein recipient heifers treated with bST (CIDR+bST) or without bST (CIDR) for synchronization. The correlation between IGF-I and P4 showed a positive pattern in the CIDR+bST group (r = 0.44, p<0.01), but a negative pattern was shown in the CIDR group (r = -0.59, p<0.02) at day 7 of estrous cycles. Although the number of recovered, transferable, and degenerated embryos was not different, quantities of grade 1 (excellent) embryos in CIDR+bST group were significantly higher than those of the CIDR group (p<0.01). The pregnancy rate was higher in the CIDR+bST recipient group compared to CIDR group (p<0.05), when the embryos were recovered from the donors treated with CIDR. However, the pregnancy was maintained highly in both recipient groups, when the embryos were produced by CIDR+bST treated donors. It can be concluded that bST administration combined with CIDR is an effective method for superovulation and synchronization treatment to stabilize plasma hormonal levels, to obtain excellent quality of embryos, and to get higher pregnancy rate.

The effect of type of vaginal insert and dose of pLH on embryo production, following fixed-time AI in a progestin-based superstimulatory protocol in Nelore cattle

The objective was to analyze and report field data focusing on the effect of type of progesterone-releasing vaginal insert and dose of pLH on embryo production, following a superstimulatory protocol involving fixed-time artificial insemination (FTAI) in Nelore cattle ( Bos taurus indicus). Donor heifers and cows ( n = 68; 136 superstimulations over 2 years) received an intravaginal, progesterone-releasing insert (CIDR ® or DIB ®, with 1.9 or 1.0 g progesterone, respectively) and 3–4 mg of estradiol benzoate (EB) i.m. at random stages of the estrous cycle. Five days later (designated Day 0), cattle were superstimulated with a total of 120–200 mg of pFSH (Folltropin-V ®), given twice daily in decreasing doses from Days 0 to 3. All cattle received two luteolytic doses of PGF2α at 08:00 and 20:00 h on Day 2 and progesterone inserts were removed at 20:00 h on Day 3 (36 h after the first PGF2α injection). Ovulation was induced with pLH (Lutropin-V ®, 12.5 or 25 mg, i.m.) at 08:00 h on Day 4 with FTAI 12, 24 and in several cases, 36 h later. Embryos were recovered on Days 11 or 12, graded and transferred to synchronous recipients. Overall, the mean (±S.E.M.) number of total ova/embryos (13.3 ± 0.8) and viable embryos (9.4 ± 0.6) and pregnancy rate (43.5%; 528/1213) did not differ among groups, but embryo viability rate (overall, 70.8%) was higher in donors with a DIB (72.3%) than a CIDR (68.3%, P = 0.007). In conclusion, the administration of pLH 12 h after progesterone removal in a progestin-based superstimulatory protocol facilitated fixed-time AI in Nelore donors, with embryo production, embryo viability and pregnancy rates after embryo transfer, comparable to published results where estrus detection and AI was done. Results suggested a possible alternative, which would eliminate the need for estrus detection in donors.

No evidence of Mycobacterium avium subsp. paratuberculosis in in vitro produced cryopreserved embryos derived from subclinically infected cows

The aim of the project was to ascertain if Mycobacterium avium subsp. paratuberculosis ( Map) could be cultured from frozen-thawed in vitro produced (IVP) embryos derived from cows with subclinical Johne's disease (JD). Straws of 109 IVP embryos were obtained from 267 cumulus–oocyte-complexes (COCs) collected from 12 clinically normal cows in which antibodies against Map were detected in blood by an enzyme-linked immunosorbent assay (ELISA). These embryos were processed, washed using the standard protocol as described by the International Embryo Transfer Society (IETS) and frozen in a commercial IVP embryo laboratory. Of the 12 donor cows, 11 had histopathological or bacteriological evidence of infection at post-mortem inspection. The frozen embryos were thawed and the contents of the straws were cultured using the radiometric mycobacterial culture method. No Map was detected in any of the 109 embryos or freezing media. This suggests that the use of in vitro produced and cryopreserved embryos derived from cows with subclinical JD poses very low, if any, risk of spreading infection to susceptible animals.

218 BREEDING TRIAL OF CONGENITAL HYDROCEPHALUS OF JAPANESE BLACK CATTLE USING EMBRYO TRANSFER

Hydrocephalus is a congenital defect of cattle. Births of Japanese Black calves with forehead doming were reported in Tohoku District from 1988 to 1989. The common finding on autopsy was the presence of excessive amounts of cerebrospinal fluid with expanding lateral ventricles without evidence of microbiologic infection. Breeding records for pedigree registration have been maintained by the Wagyu Registry Association in this area since 1960. A survey of the pedigrees suggested that an autosomal recessive gene might be responsible for the defect. The incidence of doming calves ceased by an advised mating system which excluded the suspected sires after 1990. Molecular genetics is useful for disclosing DNA sequences of a recessive gene and for detecting the carriers of the gene. This trial was conducted to produce congenital hydrocephalus calves for gene analysis using embryo transfer on a small number of the remaining cows that gave birth to affected calves over 10 years ago. One donor cow, 11 years old, was superovulated with 24 to 25 AU of FSH (Antrin; Kawasaki Mitaka K.K., Kawasaki, Japan) and inseminated with frozen semen of a suspected bull. Collected embryos were transferred on the day of the collection and the rest of them were frozen and transferred later. Superstimulation was repeated 4 times in this cow at an interval of 76-105 days during and 8-month period. A total of 4, 13, 17, and 18 embryos of good quality were collected. Eighteen fresh embryos were transferred into 7 (2 embryos each) and 4 (single embryo) recipients. Frozen embryos were transferred into 6 recipients (single embryo). Seven of 17 recipients became pregnant and gave birth to 8 calves. Three of 6 male calves born and one of 2 females showed symptoms of hydrocephalus. Clinically, 3 of the affected calves had a domed forehead and squint-eyes and were unable to stand. One calf underwent a fatal convulsion and ataxia 29 days after birth. Pathological findings of affected calves showed dilated bilateral ventricles with increased cerebrospinal fluid as typical lesions of hydrocephalus. Embryo transfer was demonstrated to be a useful tool for investigation of a potentially heritable disease. Gene scanning with microsatellite markers using the tissues from the calves and the related cattle was done. The suspected gene was found near the centromere of chromosome 3 (BTA3). It was possible to diagnose the mutant-carrier cattle using two DNA markers, INRA006 (17.0 cm) and BL41 (43.2 cm).

262 ASSESSMENT OF HSP70-1 TRANSCRIPTION LEVELS IN IMMATURE OOCYTES FROM BOS TAURUS AND BOS INDICUS COWS RAISED IN A TROPICAL CLIMATE

Heat stress is one of the main causes of low conception rate in Bos taurus cows in a tropical climate. On the other hand, in this environment, oocytes from Bos indicus show greater developmental capacity after in vitro fertilization than those from Bos taurus, suggesting an adaptation to the hot climate. Heat shock proteins (HSP) are chaperones that promote protection against heat damage, and their transcription is associated to stress. The aim of this study was to evaluate the expression of HSP70-1 gene (Genbank NM174550), a member of HSP family, in oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) cows raised in the tropical climate located at 21�35′′S latitude, 43�51′′W longitude, and 435 m altitude. Cumulus–oocyte complexes were recovered by oocyte pickup from mature non-lactating Holstein (n = 4) and Gyr (n = 4) donor cows during the hot season. Cumulus cells of viable oocytes were removed by vortexing in TALP-HEPES plus BSA, and pools (3 for each breed) with 12 immature oocytes were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction. Total RNA extraction was performed using Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and first strands were synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000; Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) and cDNA equivalent to 1.2 oocytes and gene specific primers. Expression of the GAPDH gene was used as endogenous reference. Calculations of relative quantification were performed by the comparative Ct method, using the lowest value found in Bos indicus oocytes as calibrator; values (mean � SE) are shown as n-fold difference relative to the calibrator. Statistical comparison between breeds was performed by analysis of variance. Oocytes from Holstein cows showed a higher level (P &amp;lt; 0.05) of HSP70-1 expression (1.82 � 0.22) than oocytes recovered from Gyr cows (1.12 � 0.11). Previous study reported that oocytes from Gyr cows in a tropical climate showed a higher blastocyst rate after in vitro fertilization than Holstein oocytes (Camargo et al. 2006 Reprod. Fertil. Dev. 18, 243 abst). The lower level of HSP70-1 in Gyr oocytes suggests that they were less subject to stress than the Holstein ones, which may reflect their capacity to develop after fertilization. This effect may be, at least in part, due to the ability of Bos indicus cows to regulate body temperature in a hot environment, causing less stress on oocytes. Financial support was provided by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogen�tica, Vi�osa, Brazil, for the real-time PCR machine.

84 GENE EXPRESSION DURING BLASTOCYST-TO-ELONGATED STAGE IN BOVINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

Epigenetic modification is an important factor in the development of embryos and the production of normal offspring derived from somatic cell nuclear transfer (NTSC). Several investigators have reported aberrant gene transcription in bovine NTSC embryos at the blastocyst (BC) stage. The objectives of this study were to evaluate the gene expression in NTSC embryos, which had developed to the elongated (EL) stage, and clarify differential levels of gene transcription in the embryo disc (ED) and trophectoderm (TE) of EL embryos. Five specific mRNAs [octamer-binding transcription factor (OCT-4), interferon-τ (IFN-τ), fibroblast growth factor receptor 2 (FGF-R2), and fibroblast growth factors 2 and 4 (FGF-2 and FGF-4)] were selected. Bovine BC embryos were obtained from NTSC using calf fibroblast cells or the uterus of donor cows after AI (Vivo). Some BC embryos were transferred to recipient cows at Day 7 (Day 0 = estrus), and then EL embryos were collected by uterine flushing at Day 16. Total RNA in single BC, ED, and TE were reverse-transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA was normalized to the abundance of GAPDH. A total of 15 (BC) and 7 (ED and TE) samples were used in each group to analyze the gene expression. Data on mRNA expression levels were analyzed using a Kruskal–Wallis test followed by multiple pair-wise comparisons using the Scheffe method. Most embryos (87–100%) gave positive signals of OCT-4, IFN-τ, and FGF-R2, regardless of the origin and stage of the embryos. Transcript signals of FGF-4 in BC embryos derived from Vivo (100%) and NTSC (70%) were detected with higher frequencies. At the EL stage, the FGF-4 signal was detected in only ED. The transcript of FGF-2 was detected with lower frequencies (20–27%) in BC embryos, but was consistently (71–86%) detected in ED of both groups. The relative abundance of OCT-4 expression in NTSC was higher (P &amp;lt; 0.05) than in Vivo embryos at the BC stage. In contrast, the transcript of FGF-4 at the BC stage was lower (P &amp;lt; 0.01) in NTSC than in Vivo embryos. Transcript levels of IFN-�, FGF-R2, and FGF-2 were not significantly different in both groups at the BC stage. The amount of OCT-4, FGF-4, and FGF-2 transcripts in ED were significantly (P &amp;lt; 0.05) higher than in TE. Transcript levels of these genes did not differ between NTSC and Vivo embryos. FGF-R2 levels were not significantly different in origins and tissue of EL embryos. In Vivo embryos, the IFN-τ level of TE was significantly (P &amp;lt; 0.05) higher than in ED. However, the difference in the IFN-τ transcription was not observed between ED and TE in NTSC embryos. The results of an analysis of mRNA transcripts at 2 different stages of development demonstrate that bovine NTSC embryos at the BC stage show deviations in expression patterns with respect to several genes which have important roles in cell differentiation, implantation, and embryo development, but these expressions, except for IFN-τ, were modified to a normal level according to the embryo development and differentiation.

167 EXPRESSION OF INTERFERON τ IN BOVINE EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION

Parthenogenetic activation (PA) is a useful technique for reproductive technologies such as somatic cell nuclear transfer. Furthermore, there is a possibility of embryonic stem cell establishment deriving from PA embryos (Cibelli et al. 2002 Science 295, 819). Understanding of the ability of development and differentiation in PA embryos is important for the application. This study was designed to assess the gene expression of octamer-binding transcription factor (OCT-4) and interferon τ (IFNτ) in bovine PA embryos at the blastocyst (BC) and elongated (EL) stages, and the protein secretion of IFNτ at the EL stage. PA embryos were produced from oocytes matured in vitro (24 h), activated with Ca-ionophore (5 min) and electric pulse, and then treated with cytochalasin B and cycloheximide (5 h). In vivo-produced (Vivo) embryos were obtained non-surgically at Day 8 (Day 0 = estrus) from superovulated donor cows. PA or Vivo embryos were transferred to recipient cows (PA: 10 embryos/cow; Vivo: 1 embryo/cow) at Day 8, and then recovered non-surgically with uterine flushings at Day 16. Total RNA in single BC and EL embryos were reverse transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA was normalized to the abundance of GAPDH. IFNτ secretion of uterine flushings was estimated by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). The cleavage and blastocyst developmental rates of PA oocytes were 65.8 and 29.7%, respectively. Most embryos had positive signals of OCT-4 and IFNτ regardless of the origin and stage of embryos. The relative abundance (mean � SEM) of OCT-4 expression in PA and Vivo embryos dropped to its lowest level (1.78 � 0.32 and 1.14 � 0.45, respectively) at the EL stage, and it was significantly lower than that at the BC stage (1018.87 � 148.69 and 696.29 � 151.80, respectively; P &amp;lt; 0.05). The transcript level of OCT-4 was not significantly different between PA and Vivo embryos at both stages. Although the transcript level of IFNτ in PA and Vivo embryos increased significantly at the EL stage (0.36 � 0.06 and 7.68 � 2.01, respectively) from the BC stage (0.03 � 0.01 and 0.01 � 0.004, respectively; P &amp;lt; 0.05), that in PA embryos was significantly lower than that in Vivo embryos at the EL stage (P &amp;lt; 0.01). The total amount (mean � SEM) of IFNτ in uterine flushings from cows with transferred PA embryos was 3.38 � 0.35 �g (the number of embryos in each uterine flushing was unknown), and it was low compared with that from cows with Vivo embryos (13.40 � 3.03 �g). Our results indicate that bovine PA embryos have the ability to secrete IFNτ in the uteri of recipient cows at the EL stage, and there is a similar expression pattern of OCT-4 for Vivo embryos.

412 VIABILITY OF ENZYMATICALLY ISOLATED BOVINE PRIMORDIAL AND PRIMARY OVARIAN FOLLICLES COLLECTED BY THE BIOPSY PICK-UP TECHNIQUE

Our institution recently developed a new tool for transvaginal, ultrasound-guided collection of ovarian biopsies from living donor cows (Aerts et al. 2005 Theriogenology 64, 947–957). The biopsy pickup (BPU) device consists of a modified needle guidance system, which is equipped with a trocar needle and carries a 60-cm-long true-cut biopsy needle. In a previous experiment, the presence of primordial and preantral follicles in BPU-derived biopsies was demonstrated. As follicular integrity is a prerequisite for further culture or research, the aim of the present study was to assess the viability of enzymatically isolated primordial and primary ovarian follicles collected by the BPU instrument. Four cows were subjected to a one-time BPU procedure. Four cortical biopsies were collected per ovary from each animal. Follicle viability was determined by the dual fluorescent probe technique described by Cortvrindt and Smitz (2001 Fertil. Steril. 75, 588–593). In live cells, calcein-AM is converted into calcein by intracellular esterase enzymes, producing an intense green fluorescence. In dead cells, ethidium homodimer-I is able to penetrate the damaged plasma membrane and, upon binding to nucleic acids, undergo a 40-fold enrichment of fluorescence, thereby producing an intense red signal. Upon retrieval, the biopsies were immersed in HEPES-buffered minimum essential medium (GIBCO, Grand Island, NY, USA) at 4�C. In the laboratory, the tissue samples were transferred to 50-mL conical tubes containing 20 mL PBS supplemented with 1 mg mL-1 collagenase type IA (Sigma-Aldrich NV/SA, Bornem, Belgium) and were incubated in a water bath at 37�C for 60 min. The digestion was terminated by adding an equal volume of cold PBS. The resulting suspension was centrifuged at 300g for 10 min at 8�C. The pellet was resuspended in PBS and transferred to a Petri dish. Each Petri dish was examined under a phase-contrast inverted microscope (Olympus CHX41) equipped with an eyepiece micrometer, and the primordial and primary follicles were collected with a glass micropipette. Isolated follicles were mounted on a slide in 50-�L droplets of PBS containing 2 �mol L-1 calceine-AM and 5 �mol L-1 ethidium homodimer-I (Molecular Probes, Leiden, The Netherlands), and incubated in the dark for 45 min at 37�C. After incubation, the follicles were examined under a fluorescence microscope (Olympus BX61). A total of 95 enzymatically isolated follicles were analyzed for viability with fluorescent probes; 89 (93.7%) of these were viable. Viable follicles were retrieved from all animals. This experiment confirms that viable primordial and primary follicles can be retrieved from living donor cows by the BPU procedure, which makes the technique suited for further culture or research.

301 EFFECT OF CO-CULTURE WITH DIFFERENT STAGE EMBRYOS ON DEVELOPMENT OF ALGINATE-ENCAPSULATED SMALL NUMBER BOVINE EMBRYOS

Previous studies show that embryos cultured in large numbers have better developmental competence than those in small numbers in mice, sheep, and cattle. We have reported that co-culture of bovine embryos encapsulated in calcium-alginate gel (microcapsule) improves the development of embryos cultured in small numbers (Kobayashi et al. 2006 Reprod. Fertil. Devel. 18, 248). This method is beneficial for culture of small numbers of embryos such as OPU-derived embryos by recognizing the individual donor cows with abattoir-derived unidentified IVF embryos. In the previous study, we used the same stage embryos for co-culture of encapsulated embryos. However, in the case of unavailability of the same stage embryos, encapsulated embryos may be co-cultured with different stage embryos. Effect of different stage embryos on co-culture of encapsulated embryos is not clear. In the present study, we investigated the effect of co-culture of different stage embryos on development of encapsulated small number embryos. In vitro-matured and fertilized zygotes from abattoir derived ovaries were used for the experiment. Small numbers of zygotes were encapsulated by alginate-gel microcapsule to distinguish from co-cultured embryos. Encapsulation was carried out by putting the 1% sodium alginate solution containing zygotes slowly into 0.1% calcium chloride solution (microcapsule). The embryos used for co-culture were produced by IVF 1-3 days before preparation of encapsulated zygotes (Day 1, Day 2, and Day 3). Five encapsulated zygotes were cultured with 15 embryos for co-culture in one droplet (100 �L) made by CR1aa + 5% CS, at 38.5�C, CO2 in air. Encapsulated zygotes co-cultured with the same stage of zygotes were assigned as a control (Day 0). The rates of cleavage on Day 2 and development to blastocyst stage on Day 9 were recorded. Data were analyzed by Student&amp;apos;s t-test. No significant difference was observed in the rate of cleavage in all experimental groups compared with control (Day 1: 72.5&amp;percnt; (n &amp;equals; 80) vs. control: 75.7&amp;percnt; (n &amp;equals; 70); Day 2: 76.3&amp;percnt; (n &amp;equals; 80) vs. control: 82.5&amp;percnt; (n &amp;equals; 80); and Day 3: 78.7&amp;percnt; (n &amp;equals; 75) vs. control: 70.8&amp;percnt; (n &amp;equals; 65). There was not a significant difference in the rate of development to the blastocyst stage in all experimental groups compared with control (Day 1: 42.5&amp;percnt; vs. control: 44.3&amp;percnt;; Day 2: 43.8&amp;percnt; vs. control: 38.8&amp;percnt;; Day 3: 44.0&amp;percnt; vs. control: 35.4&amp;percnt;). These results indicate that co-culture of different stages of embryos can normally support the development of small numbers of encapsulated embryos. These methods are useful to improve the development of small numbers of embryos derived from OPU-IVF embryos without synchronization of the developmental stage of co-cultured embryos.

Measures of Maternal-Fetal Interaction in Day-30 Bovine Pregnancies Derived from Nuclear Transfer

Embryonic mortality and abnormal placental morphology have been reported by most researchers studying nuclear transfer (NT), and it now is accepted that placental anomalies and poor development of cloned embryos are related. As early as day 50 of gestation, cloned bovine concepti exhibit poor structural organization of the developing placentomes. These experiments were designed to identify alterations in maternal-fetal interactions during establishment of the placentas of NT-derived embryos at day 30 of gestation. Bovine NT embryos were produced using cultured fibroblast cells from a single Hereford donor cow, and control embryos were derived from in vitro fertilization (IVF). Following in vivo culture in ligated sheep oviducts, day-8 blastocysts were transferred to synchronized recipient heifers. Tissues recovered from viable day-30 pregnancies were analyzed by real-time RT-PCR, immunohistochemistry, and quantitative histological techniques. Immunoperoxidase staining of caruncular tissue from NT- and IVF-derived pregnancies revealed no significant differences in expression of the extracellular matrix proteins, collagen type IV and laminin, or the receptor subunits, integrins alpha1 and alpha3, suggesting that altered expression of these proteins at day 30 of gestation is not a primary cause of abnormal placentome structure in cloned concepti. Percentage of binucleate cells (BNC) within the trophoblast also was similar in NT- and IVF-derived pregnancies; however, expression of the BNC-specific placental lactogen (PL) transcript was elevated in NT-derived concepti (p < 0.05). These results indicate that regulation of PL transcription was altered in cloned day-30 placental tissues, suggesting the presence of irregular fetal-maternal signaling patterns that might undermine continued development of NT-derived concepti.

The timing of ovulation and insemination schedules in superstimulated cattle

The development of treatments that control follicular wave dynamics during the bovine estrous cycle has resulted in interesting possibilities for the precise control of follicular wave emergence and the time of ovulation. For superstimulation, follicular wave emergence can be controlled by ultrasound-guided follicle ablation with FSH treatments initiated 1 or 2 d later, or injection of estradiol combined with progesterone at the time of insertion of a progestogen releasing device and FSH treatments beginning 4 d later. These are the most widely used protocols for superstimulation of donor cows because they offer the convenience of being able to initiate treatments quickly and at a self-appointed time, without reducing the number of transferable embryos. However, these protocols still require precise estrus detection of donors following superstimulation in order to conduct AI at the most appropriate time. Recent studies have been designed to develop superstimulation protocols that involve fixed-time AI of donors, without regard to estrus detection. Results presented herein indicate that delaying the removal of a progestogen releasing device, combined with the administration of GnRH or porcine LH (pLH) 12 or 24 h later results in predictable, synchronous ovulations, permitting fixed-time AI without reducing the numbers or quality of embryos. These protocols facilitate the application of on-farm embryo transfer programs because they are practical, easy to administer by farm personnel, and more importantly, they eliminate the need for detecting estrus.

Ovum Pick Up and In Vitro Production in the bovine after use in several generations: A 2005 status

The first In Vitro Produced (IVP) calf was born in 1981 and the non-surgical Ovum Pick Up (OPU) technique for the bovine was adapted from the human in 1987. Since then, considerable research has been aimed at improving both technologies in the bovine. Both OPU and IVP can now be seen as mature technologies. It can be estimated that more than 200,000 IVP calves have been born world wide to date, and when the two technologies are combined they are capable of producing over 50 calves per donor cow per year, albeit with a large variation between donors. Not many new breakthroughs are expected for OPU. For IVP however, automation and miniaturization as well as a greater understanding of the embryo through the application of gene based technologies such as micro-arrays, may provide an in vitro environment that is more in vivo-like than traditional micro drop/well systems. This improved environment should result in higher embryo developmental rates as well as improved quality and welfare of subsequent offspring. The application of OPU/IVP has progressed from treating infertile high genetic multiple ovulation and embryo transfer (MOET) cows in commercial situations to enhancing breeding scheme designs. With the bovine genome being rapidly sequenced and bovine genes for traits of economic interest becoming available in the coming years, OPU/IVP will prove invaluable in rapidly multiplying rare genes or Quantitative Trait Loci (QTL) of high value. In due course, it is anticipated that Marker Assisted Selection or Gene Assisted Selection (MAS/GAS) schemes will be more widely implemented. In addition, OPU, and particularly IVP, provide the basis for more advanced technologies such as cloning and transgenics. This paper is dedicated to celebrate and recognize the significant contributions made by Theo Kruip (1939–2003) to the wide area of bovine OPU and IVP.

In vitro production of bovine embryos using sex-sorted sperm

The objective of this study was to investigate the suitability of sex-sorted sperm for producing viable in vitro embryos for subsequent transfer into recipient cows and heifers on commercial dairy farms. From August 2002 to June 2003, ovaries were collected from 104 producer-nominated Holstein donor cows on seven Wisconsin farms via colpotomy or at slaughter. Oocytes ( N = 3526) were aspirated from these ovaries, fertilized 22 ± 0.2 h later, and cultured to the morula or blastocyst stage. The fluorescence-activated cell sorting (“Beltsville”) approach was used to produce (primarily) X-bearing sperm from the ejaculates of three young Holstein sires, and 365 transferable embryos were produced. On average, 3.6 ± 0.3 (means ± S.E.M.) transferable embryos were produced per donor, including 1.4 ± 0.2 (Grade 1), 1.5 ± 0.2 (Grade 2), and 0.7 ± 0.1 (Grade 3) embryos. Number of usable oocytes per donor (33.9 ± 3.3) and percent cleavage (51.1 ± 1.9) were significant predictors of the number of blastocysts that developed. Mean conception rates for the resulting in vitro embryos were 34.2 ± 1.6% in yearling heifer recipients and 18.2 ± 0.7% in lactating cow recipients. Additional oocytes ( N = 3312) from ovaries of anonymous donors ( N unknown) collected at a commercial abattoir were fertilized using unsorted sperm, and the percentage of these that developed to blastocyst stage (20.1 ± 2.9) was greater ( P < 0.05) than the corresponding percentage (12.2 ± 2.3) achieved with sex-sorted sperm using oocytes ( N = 1577) from the same source. In summary, we inferred that in vitro embryo production may be a promising application of sex-sorted sperm in dairy cattle breeding, but that the biological causes of impaired embryo development in vitro and compromised conception rates of transferred embryos should be further investigated.

In Vitro Embryo Production: Growth Performance, Feed Efficiency, and Hematological, Metabolic, and Endocrine Status in Calves

The potential management benefits of in vitro embryo production have been offset by an increased incidence of health-related problems in resulting calves [increased birth weight, congenital abnormalities, and peri- and postnatal mortality (large-offspring syndrome)] and of recipient cows (prolonged gestation, dystocia, increased hydroallantois, abortion). The aim of the present research was to determine whether relevant metabolic, endocrine, or hematological traits could be related to the causes of enhanced growth performance of in vitro fertilized calves. Growth performance and feed efficiency as well as hematological, metabolic, and endocrine traits studied in calves derived from in vitro-produced embryos (IVP; n=11) and in calves derived from artificial insemination (AI; n=8). Donor cows from which oocytes for in vitro fertilization were obtained had a heterogeneous background, thus excluding genetic maternal influences. On the other hand, semen for in vitro fertilization and for artificial insemination was from the same bull, and recipient cows were held under the same husbandry and feeding conditions as AI cows, thus reducing the variability. Blood samples were collected preprandially on d 1, 2, 3, 4, 7, 14, 28, 56, and 112 of life and every 20min between 0830 and 1630h on d 7 and 112 for the evaluation of growth hormone secretory patterns. Gestation of IVP cows was longer than that of AI cows, but birth weights were similar in both groups. Feed intake, average daily gain, and body length during the experimental period, body weight from wk 8 to 16, and gain/feed ratio during the first month of life were higher in IVP than in AI calves. At birth, potassium, 3,5,3′-triiodothyronine, and thyroxine concentrations were lower in IVP than in AI calves. Concentrations of sodium and potassium on d 7, of triglycerides on d 28, and of albumin on d 56 were higher in IVP than in AI calves. In conclusion, IVP calves had higher feed intake and growth rate during the entire growth period and improved feed efficiency in the first month of life than AI calves, but this was not mirrored by consistent changes of hematological, metabolic, or endocrine traits, whose concentrations were in the normal range. Additional work is needed to study IVP calves under field conditions.

The usefulness of a single measurement of insulin-like growth factor-1 as a predictor of embryo yield and pregnancy rates in a bovine MOET program

The objective was to determine if a single measurement of plasma insulin-like growth factor-1 (IGF-1) could predict the number of viable embryos obtained from donors and the likelihood of pregnancy in recipients in multiple ovulation and embryo transfer (MOET) programs in cattle. The embryo yields from 101 embryo recoveries were examined in maiden Holstein heifers ( n = 75) and multiparous Holstein cows (lactating cows n = 20, dry cows n = 6). Donors were superstimulated with FSH and embryo recovery was done non-surgically 7 days after artificial insemination. Embryos were classified according to the IETS criteria. Pregnancy rates in 100 maiden Holstein heifer recipients were analysed. Recipients were on day 7 ± 1 of the estrous cycle at transfer. Pregnancy diagnosis was carried out at day 30 (PD30) and rechecked at day 60 (PD60) after transfer. Blood samples from coccygeal vessels taken at the time of embryo recovery (donors) and transfer (recipients) were analysed for IGF-1, insulin, β-hydroxybutyrate (β-OHB), non-esterified fatty acids (NEFA), urea and cholesterol. There was a negative correlation between the number of viable embryos and insulin ( r = −0.33, P = 0.025) in donor heifers. In donor cows, the number of viable embryos was correlated with IGF-1 ( r = 0.43, P = 0.028) and cholesterol ( r = −0.43, P = 0.027). In recipients, PD30 and PD60 were not affected by any of the circulating parameters analysed. Insulin, IGF-1 and cholesterol only explained 8.9, 13.9 and 15.8% of the variation in the production of viable embryos, respectively. Several factors affect MOET programs and under the circumstances of the present study the usefulness of hormonal and metabolic profiles as predictors of the outcome of this biotechnology was limited.

Long-Term Bovine Hematopoietic Engraftment with Clone-Derived Stem Cells

Therapeutic cloning by somatic cell nuclear transfer offers potential for treatment of a wide range of degenerative disease. Nuclear transplantation with neo (r)-marked somatic nuclei from 10-13-year-old cows was used to generate cloned bovine fetuses. Clone fetal liver (FL) hematopoietic stem cells (HSC) were transplanted into two busulfan-treated and one untreated nuclear donor cows. Hematopoiesis was monitored over 13-16 months by in vitro progenitor and HSC assays. Chimerism was demonstrated by PCR in blood, marrow, lymph nodes, and endothelium, peaking at levels of 9-17% in blood granulocytes but at lower levels in lymphocyte subsets (0.1-0.01%). Circulating progenitors showed high levels of chimerism (up to 60% neo (r+)) with persisting fetal features. At sacrifice, the animal that had no pre-transplant myelosupression showed persisting donor cells in blood and lymph nodes, and in marrow 0.25% of progenitor cells and a detectable fraction of stem cells were neo (r+). The fetal HSC showed a 10-fold competition advantage over adult HSC. Cloning generated histocompatible HSC capable of long-term multilineage engraftment in a large animal model.