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Direct transfer (DT) of cryopreserved embryos to recipients facilitates on-farm application. We analyzed a new freezing/thawing (F/T) procedure for in vitro produced (IVP) embryos, integrating: 1) an ethylene-glycol based system; 2) a culture step without protein; and 3) a synthetic protein substitute (CRYO3) in cryopreservation medium. IVP embryos from abattoir ovaries were cultured in groups in BSA-containing synthetic oviduct fluid with or without 0.1% fetal calf serum (FCS) until Day-6. Morulae and early blastocysts were subsequently cultured without protein from Day-6 onwards. Day 7 and Day 8 expanded blastocysts (EXB) were subjected to F/T or vitrification/warming (V/W). Thawed and warmed EXB were cultured in vitro , and development rates, cell counts and dead cells were analyzed in surviving embryos. V/W improved survival over F/T (live and hatching rates at 2 h, 24 h and 48 h) (P < 0.0001), and FCS before Day 6 did not affect in vitro survival. After F/T, embryos had lower cell counts in the ICM, TE and total cells than after V/W. Day-7 embryos after F/T showed % apoptotic, % pycnotic and % total dead cells higher (p < 0.05) than their Day-8 counterparts, probably because F/T reduced the numbers of ICM cells within Day-8 embryos. Thereafter, Day-7 blastocysts were transferred to heifers in an experimental herd. There were no differences in birth rates with frozen (-FCS [n = 40]: 45%; +FCS [n = 14]: 28%), vitrified (-FCS [n = 47]: 53%; +FCS [n = 11]: 36%) and fresh (-FCS [n = 30]: 47%; +FCS [n = 17]: 53%) embryos. However, frozen embryos produced with FCS showed 5/9 miscarriages after Day-40. Calves born from frozen (n = 22), vitrified (n = 29) and fresh (n = 22) transfers did not differ in birth weight, gestation length and daily gain weight (P > 0.10). Subsequently, transfer of frozen embryos (n = 29) derived from oocytes collected from live, hormonally stimulated cows in experimental herd, led to pregnancy rates of 57% (heifers) and 40% (dry cows). with EXB on Day-62 Finally, embryos produced with BSA were transferred to cows in an on-field trial (frozen [n = 80]; fresh [n = 58]), with no differences in pregnancy rates (days 30–40). Pregnancy and birth rates could not be predicted from in vitro approaches. The new F/T system yields pregnancy and birth rates comparable to vitrified and fresh embryos without birth overweight. The absence of products of animal origin, defined chemical composition, and direct transfer entail sanitary, manufacturing and application advantages. • Bovine IVP embryos frozen in chemically defined media lead to birth rates comparable to vitrified and fresh embryos. • Pregnancy and birth rates are not predicted from in vitro experiments. • Cryopreservation of embryos developed in a 24h single culture step without protein does not induce birth overweight. [ABSTRACT FROM AUTHOR]

Objectives were to determine effects of: 1) handling temperament and administration of flunixin meglumine, an inhibitor of prostaglandin F2a (PGF2a) synthesis, given at the time of embryo transfer, on pregnancy rates in beef cattle embryo transfer recipients; 2) handling temperament and flunixin meglumine on peripheral concentrations of progesterone, cortisol, substance-P, prostaglandin F metabolites (PGFM, (13,14-dihydro-15-keto-PGF2a) and isoprostane 8-epi PGF2a; and 3) flunixin meglumine treatment on proportion of non-pregnant recipient cows returning to estrus within an expected interval. Angus cross beef cows (n = 710) at 7 locations were assigned a body condition score (BCS: 1, emaciated; 9, obese) and a temperament score [0, calm, slow chute exit; walk (n = 352), 1, excited, fast chute exit; jump, trot or run (n = 358)] and were synchronized with Select-Synch with a controlled internal drug release (CIDR) protocol. Estrus detection aids were applied at CIDR removal and cows were observed thrice daily for estrus until 72 h. Recipient cows that expressed estrus and had a corpus luteum received a frozen-thawed embryo on Day 7 after estrus. At the time of transfer, recipient cows were randomly allocated to receive 10 mL of flunixin meglumine im, immediately after transfer (n = 365) or served as an untreated control (n = 345). In a subset of cows (n = 80), ovarian ultrasonography was performed on the day of embryo transfer to determine corpus luteum volume and blood samples were collected twice, at the time of embryo transfer and 7 d later. All cows received estrus detection aids again on Day 14 (7 d after embryo transfer) and were observed for estrus twice daily until Day 24. Accounting for treatment (P > 0.1), embryo transfer difficulty score (P < 0.1), temperament by treatment interaction (P < 0.05), recipient cows with calm temperament had a higher pregnancy rate compared to those with an excited temperament [59.4 (209/352) vs 51.7% (185/358)]. The pregnancy rate for excitable cows without flunixin meglumine was lower (46.3% 81/175) compared to excitable cows that did received flunixin meglumine [56.8% (104/183)], and calm cows that did [59.3% (108/182)] or did not [59.4% (104/170)] receive flunixin meglumine. Proportions of non-pregnant recipient cows returning to estrus on Days 18–24 were not different between flunixin meglumine and control groups, 87.6% (134/153) and 84.0% (137/163), respectively (P > 0.1). At the time of embryo transfer and 7 d later, there were moderate to strong correlations among circulating concentrations of progesterone, cortisol, substance-P, PGFM and isoprostane 8-epi PGF2a. Among excitable cows, progesterone concentrations were lower and cortisol, substance-P, PGFM and isoprostane 8-epi PGF2a concentrations were greater for cows in the control group compared to cows that received flunixin meglumine. In conclusion, administration of flunixin meglumine improved pregnancy rates in excitable recipient cows following embryo transfer without affecting the proportion of non-pregnant cows returning to estrus. [ABSTRACT FROM AUTHOR]

Two commercial FSH products were compared in a retrospective study on 3990 commercial superovulations and embryo recoveries in dairy heifers and cows. In addition, the 56-day nonreturn rate of 19,400 embryos produced with these two preparations was analyzed. Embryo collections were performed during a 16-year period from donors of Holstein and Ayrshire breeds. Folltropin (Vetoquinol S.A., Lure cedex, France) group (Group F) consisted of 2592 superovulations, of which 80% were performed on heifers and 20% on cows, and Pluset (Laboratorios Calier, S.A., Barcelona, Spain) group (Group P) of 1398 treatments, of which 66% and 34% were on heifers and cows, respectively. Total number of recovered structures, number of transferable embryos, and the proportion of unfertilized ova (UFO) and degenerated embryos were analyzed. Distribution of embryos into quality grades (1–3) and developmental stages (4–9) according to the IETS classification guidelines and means for each collection were evaluated. The proportion of low-responders having fewer than five corpora lutea and yielding fewer than five embryos or ova was investigated for each treatment. Group P yielded 1.1 recovered structures more than Group F (P < 0.001). Consequently, however, the number of transferable embryos did not differ among the groups, being 7.0 and 7.1 in Groups F and P, respectively. Instead, there was an increase in the number of UFO from 2.0 in Group F to 3.0 in Group P (P < 0.001). The quality of embryos and the developmental stages were similar between the groups and there was no difference in the proportion of low-responding donors in Group F and Group P. Also, there was no difference in the nonreturn rate after transfer of embryos originating from donors superovulated with Folltropin or Pluset. It was concluded that equal numbers of transferable embryos and pregnancies can be achieved with Folltropin and Pluset. [ABSTRACT FROM AUTHOR]

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo -derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8–16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 10 7 Chlamydia /mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO 2 , the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus . The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at −20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP. [ABSTRACT FROM AUTHOR]

This study aimed to assess the suitability of luteal blood flow analyses measured by color Doppler ultrasonography (CDUS), to predict pregnancy at pre- and post-embryo transfer (ET) in dairy cows, and to compare with the established criterion like luteal size and plasma progesterone (P 4 ) concentrations. Lactating Holstein cows (n = 65) with spontaneous (n = 34) or synchronized estrus (n = 31) were examined. Cows with a CL greater than or equal to 20 mm in diameter (n = 58) received embryo transfer on Day 7 (Day 0 = estrus). Brightness mode images were captured for calculation of the CL area, luteal cavity area, and dominant follicle area on Days 3, 5, 7, and 14. Color Doppler ultrasonography examinations were conducted to determine the blood flow area (BFA) within the CL at the maximum diameter and the time-averaged maximum velocity (TAMV) of the base of the spiral artery on the same days. Plasma P 4 concentrations were determined from blood samples collected at each ultrasound examination. Pregnancy was diagnosed by an ultrasound on Day 30. There was no significant difference in the proportion of cows received embryo (91.2% vs. 87.1%, P = 0.70) and pregnancy rate (58.1% vs. 59.3%, P = 1.00) between the spontaneous estrus and synchronized groups. The BFA values of the pregnant group (n = 34) were approximately 1.42 and 1.54 times higher than those of the nonpregnant group (n = 24) on Days 7 (0.54 ± 0.04 cm 2 vs. 0.38 ± 0.02 cm 2 ; P < 0.01) and 14 (0.80 ± 0.23 cm 2 vs. 0.52 ± 0.22 cm 2 ; P < 0.01), respectively. The TAMV of the pregnant group was approximately 1.45 times higher than that of the nonpregnant group on Day 14 (57.8 ± 3.5 cm/s vs. 40.0 ± 3.3 cm/s; P < 0.01). However, no differences were found in the CL area, CL tissue area, dominant follicle area, and plasma P 4 concentrations among these groups. In addition, the best logistic regression model to predict pregnancy included scores for BFA on Day 7, BFA and TAMV on Day 14. Setting the cutoff value of BFA at 0.43 cm 2 yielded the highest sensitivity (79.4%) and specificity (75.0%) on Day 7, indicating the effectiveness of using BFA data for predicting pregnancy on Day 7. Furthermore, setting the cutoff value at one obtained from a sample with BFA 0.63 cm 2 and TAMV 50.60 cm/s yielded the highest sensitivity (85.3%) and specificity (91.7%) on Day 14. In conclusion, the evaluations of BFA on Day 7, and paired BFA and TAMV on Day 14 represent reliable predictors of pregnancy in the cow. [ABSTRACT FROM AUTHOR]

Artificial insemination and in vitro embryo production are powerful tools for disseminating superior genetic qualities and improving the reproductive performance of dairy and beef cattle. In conjunction with these biotechnologies, sexed-sorted semen has been used to obtain offspring of a predetermined sex. This study compared the pregnancy rates obtained using in vitro fertilization/timed embryo transfer (IVF/TET) and timed artificial insemination (TAI), both performed using sexed-sorted (Y-chromosome-bearing) semen obtained from the same bull. For the in vitro embryo production, the ovaries of 250 Nelore cows with known histories were collected in the slaughterhouse and used for IVF. After evaluation of the recipients (IVF/TET group; n = 974), the resultant embryos were transferred to the females with corpus luteum (n = 822). The pregnancy-related data for this group were compared with those for the TAI group (n = 974). Ultrasonography was performed at 60 days to determine the pregnancy status and confirm the sex of the fetus. A total of 2008 oocytes produced 1050 embryos, with 52% of them reaching the blastocyst stage. The pregnancy rate and the accuracy in determining the fetal sex were 35.4% (345/974) and 95.07% (328/345), respectively, for the IVF/TET group and 30% (293/974; P < 0.05) and 94.88% (278/293), respectively, for the TAI group. In the present study, we concluded that male calves could be better obtained using IVF/TET rather than TAI; therefore, this strategy can be considered to increase the pregnancy rate of beef cattle. [ABSTRACT FROM AUTHOR]

The gross anatomic features (cotyledonary type) and histologic classification (synepitheliochorial) of the bovine placenta have been known for many years. Thorough ultrastructural analysis as well as a variety of descriptive studies dealing with the localization of cytoskeletal filaments, extracellular matrix, growth factor systems, steroid hormone receptors, and major histocompatibility complex have contributed further significant knowledge. However, this knowledge was not sufficient to solve clinical placenta-based problems, such as retained fetal membranes. Owing to the complexity of the fetomaternal interface in vitro , culture systems have been developed. As trophoblast giant cells (TGC) are thought to be key players in the cattle placenta, most cell culture models attempt to overcome the pitfall of losing the entire TGC population in vitro . Nevertheless, distinct cell line–based in vitro systems such as cell monolayers or 3-dimensional (co-culture) spheroids were generated for the fetal (trophoblast) and maternal (uterine epithelium) placental compartments. Monolayers have been used to study for example, growth factor or hormonal signaling and TGC formation, whereas spheroids served as models for, for example, trophoblast attachment, uterine epithelium depolarization, and also TGC formation. In the future, the use of more improved culture models might lead to better treatments of retained fetal membranes and increased prevention of embryonic loss. In addition, the in vitro models could shed more light on the mechanisms of the differentiation of uninucleate trophoblast into TGC. [ABSTRACT FROM AUTHOR]

The embryo transfer techniques used in small ruminants worldwide are based in surgical procedures. These actions are performed under general anesthesia which needs a combination of animal fasting and drugs for secure animal handling and surgery manipulations. Therefore, it involves risks to animal health and life. The major limiting sequels are adhesions formed by the abdominal surgery, in the ovaries, uterus, or between them. These occurrences can both compromise uterus accessing and oocyte capture and are responsible for decreasing success and limiting successive embryo collections. In contrast, nonsurgical embryo procedures can be performed in a relatively simplified way. Nonsurgical embryo recovery does not need animal prolonged starvation, drug retention is minimized, and donors can stay in a standing position. After the end of embryo recovery, donors are promptly restored to their routine housing and feeding. Furthermore, this technique does not need incisions and, therefore, can be used repetitively in superovulated or nonsuperovulated goats and sheep for embryo recovery—a similar procedure done in cattle. In Brazil, promising results are reported using nonsurgical embryo transfer in recipient goats, and studies are currently evaluating similar procedures in sheep. Therefore, this review aimed to present the current panorama of nonsurgical embryo transfer in sheep and goats. [ABSTRACT FROM AUTHOR]

The objective of the present study was to examine whether genomic selection of females interacts with the use of reproductive technologies (RT) to increase annual monetary genetic gain (AMGG). This was tested using a factorial design with 3 factors: genomic selection of females (0 or 2,000 genotyped heifers per year), RT (0 or 50 donors selected at 14 mo of age for producing 10 offspring), and 2 reliabilities of genomic prediction. In addition, different strategies for use of RT and how strategies interact with the reliability of genomic prediction were investigated using stochastic simulation by varying (1) number of donors (25, 50, 100, 200), (2) number of calves born per donor (10 or 20), (3) age of donor (2 or 14 mo), and (4) number of sires (25, 50, 100, 200). In total, 72 different breeding schemes were investigated. The profitability of the different breeding strategies was evaluated by deterministic simulation by varying the costs of a born calf with reproductive technologies at levels of €500, €1,000, and €1,500. The results confirm our hypothesis that combining genomic selection of females with use of RT increases AMGG more than in a reference scheme without genomic selection in females. When the reliability of genomic prediction is high, the effect on rate of inbreeding (ΔF) is small. The study also demonstrates favorable interaction effects between the components of the breeder's equation (selection intensity, selection accuracy, generation interval) for the bull dam donor path, leading to higher AMGG. Increasing the donor program and number of born calves to achieve higher AMGG is associated with the undesirable effect of increased ΔF. This can be alleviated, however, by increasing the numbers of sires without compromising AMGG remarkably. For the major part of the investigated donor schemes, the investment in RT is profitable in dairy cattle populations, even at high levels of costs for RT. [ABSTRACT FROM AUTHOR]

This study investigated the pregnancy rate and calf mortality after transfer of embryos produced using sex-sorted semen. Data for 12,438 embryo transfers performed on dairy farms were analyzed. Of these, 10,697 embryos were produced using conventional semen (CONV embryos) and 1741 using sex-sorted semen from 97 bulls (SEX embryos), predominantly of Ayrshire and Holstein breeds. Of the CONV embryos, 27.4% were transferred fresh, whereas of the SEX embryos, 55.7% were fresh. Recipient attributes (breed, parity, number of previous breeding attempts, and interval from calving to transfer) were comparable for both embryo types, heifers representing 57.8% of recipients in the CONV group and 54.8% in the SEX group. Recipients that were not artificially inseminated or did not undergo a new embryo transfer after the initial embryo transfer and had registered calving in fewer than 290 days after the transfer were considered pregnant. Pregnancy rate for recipients receiving CONV embryos was 44.1%, and for those receiving SEX embryos, it was 38.8%. The odds ratio for pregnancy in recipients receiving CONV embryos was 1.34 compared with SEX embryos (P < 0.001). The proportion of female calves was 49.6% and 92.3% in CONV and SEX groups, respectively. Overall, calf mortality was comparable in both groups. Mortality was similar in CONV and SEX groups (6.6% and 7.7%, respectively) for female calves. For male calves, mortality was 9.2% in the CONV group but significantly higher, 16.0% (P < 0.05), in the SEX group. This study showed that transfer of embryos produced with sex-sorted semen decreased the pregnancy rate by about 12% compared with embryos produced using conventional semen. Mortality of male calves born from SEX embryos was higher than for those born from CONV embryos. [ABSTRACT FROM AUTHOR]

The hormone progesterone is essential for proper embryonic development. The objective of this study was to examine the relationship between recipient serum concentrations of progesterone, at the time of embryo transfer and at conceptus recovery, on conceptus development from in vivo- or in vitro-produced embryos. Embryos were produced in vivo by superovulation of Holstein cows (IVO; n = 17) or in vitro with either serum-containing (IVPS; n = 27) or serum-restricted medium (IVPSR; n = 34). Single grade I blastocysts from each embryo production system were transferred into heifers on day 7 of development. Conceptuses were recovered on day 17 of gestation and classified as complete, degenerated, or no conceptus. Compared with the IVO group, in vitro-produced embryos had more ( P = 0.055) degenerated conceptuses (IVO, 0%; IVPS, 18.5%; and IVPSR, 20.6%). There were no differences in progesterone concentrations at the time of transfer when recipients received either male or female embryos ( P > 0.05). Progesterone concentrations in recipients receiving in vivo-produced embryos were higher ( P < 0.05; 3.74 ± 0.4 ng/mL; least-squares mean ± standard error of the mean) on day 7 compared with those receiving in vitro-produced embryos (IVPS, 2.4 ± 0.2; IVPSR, 2.58 ± 0.3 ng/mL). However, there was no difference in progesterone concentration on day 7 between treatment groups for heifers from which short conceptuses (≤194 mm) were recovered on day 17. In contrast, when longer (>194 mm) conceptuses were recovered on day 17, heifers receiving in vitro-produced embryos had lower ( P = 0.05) serum concentrations of progesterone on day 7 compared with those receiving in vivo-produced embryos (IVPS, 2.2 ± 0.5; IVPSR, 2.3 ± 0.5; IVO, 3.9 ± 0.5 ng/mL). In conclusion, differences in autonomy may exist between in vitro- and in vivo-produced embryos during the period of conceptus elongation with in vitro-produced embryos relying more on intrinsic factors to influence elongation. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the impact of varying features of an open multiple ovulation and embryo transfer (MOET) nucleus used in a genomic dairy cattle breeding scheme on both genetic gain and inbreeding rates. The Viking Red breeding scheme served as a case study to design scenarios that were stochastically simulated. We analyzed the number of AI sires used in the breeding population, the number of flushed heifers, the number of flushings per heifer and the genotyping capacity allocated to young females. The results supported that setting up a MOET program in a genomic dairy cattle scheme increases genetic gain without increasing inbreeding rates when the MOET nucleus size and the number of AI sires in service are large enough. Secondly, it was shown that increasing the number of genotyped heifers could not compensate the loss in genetic gain caused by closing the MOET nucleus. On the contrary, when extending the flushing capacity of the MOET program, increasing the number of flushings per heifer had a greater impact on genetic gain, but also on inbreeding rates, than increasing the number of flushed females. So, when a constraint applies on the flushing capacity in an open MOET scheme and the achieved inbreeding rate permits it, it seems more relevant to increase the number of flushings per heifer than the number of flushed heifers. Results also indicated that the number of genotypings allocated to females had to be sufficient to get maximal returns from the MOET scheme. In this case study, little extra genetic gain could be obtained by extending the MOET scheme size with the initial genotyping strategy (800 genotyped females). Indeed, the genotyping capacity should permit to genotype all heifers produced in the MOET scheme to discriminate the best heifers within families, and should be also sufficient to identify the best heifers outside the MOET nucleus. [ABSTRACT FROM AUTHOR]

Abstract: After the first successful transfer of mammalian embryos in 1890, it was approximately 60 years before significant progress was reported in the basic technology of embryo transfer (ET) in cattle. Starting in the early 1970s, technology had progressed sufficiently to support the founding of commercial ET programs in several countries. Today, well-established and reliable techniques involving superovulation, embryo recovery and transfer, cryopreservation, and IVF are utilized worldwide in hundreds, if not thousands, of commercial businesses located in many countries. The mean number of embryos produced via superovulation has changed little in 40 years, but there have been improvements in synchrony and hormonal protocols. Cryopreservation of in vivo-derived embryos is a reliable procedure, but improvements are needed for biopsied and in vitro-derived embryos. High pregnancy rates are achieved when good quality embryos are transferred into suitable recipients and low pregnancy rates are often owing to problems in recipient management and not technology per se. In the future, unanticipated disease outbreaks and the ever-changing economics of cattle and milk prices will continue to influence the ET industry. The issue of abnormal pregnancies involving in vitro embryos has not been satisfactorily resolved and the involvement of abnormal epigenetics associate with this technology merits continued research. Last, genomic testing of bovine embryos is likely to be available in the foreseeable future. This may markedly decrease the number of embryos that are actually transferred and stimulate the evolution of more sophisticated ET businesses. [Copyright &y& Elsevier]

Abstract: The insulin-like growth factor 1 receptor (IGF1R) is a membrane glycoprotein mediating most biological actions of IGF1 and IGF2, and has an important effect on ovulation, pre-implantation embryo development and pregnancy rate. The objectives of this study were to detect IGF1R gene polymorphisms of cattle and analyze the relationship with superovulation performance and pregnancy rates after embryo transfer (ET), as well as the hormone concentrations at the day of ET. One reported SNP of IGF1R G404T and a novel SNP of IGF1R G399A were analyzed in 170 Chinese Holstein donor cows and 118 Luxi recipients cattle. Statistical analysis revealed that the G404T mutation was associated (p =0.019) with increased ovulation rate and females with this mutation had enhanced performance in producing transferable embryos. For the polymorphic locus G399A, recipients with g.399 GG and g.399 GA genotypes had greater pregnancy rates after ET than that of g.399 AA genotype. Furthermore, the same tendency was observed that the genotype groups with greater pregnancy rates had greater progesterone and lesser estrogen concentrations, but these did not reach statistical significance. Results of the present study showed, for the first time, that the polymorphism in IGF1R is associated with superovulation traits, and indicated that the IGFIR gene can be used as a potential marker for donor selection. [Copyright &y& Elsevier]

Abstract: It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 μM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4–0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos. [Copyright &y& Elsevier]

Abstract: The first successful equine embryo transfer was reported in 1972, 21 years after the first reported embryo transfer in cattle. Adaptations of embryo transfer and the various related technologies have generally been more rapid in the equine than in cattle, with the exceptions of superovulation, in vitro fertilization and cryopreservation. Recent progress has been achieved in all three of these areas. This paper presents a time line of various events in the history of equine embryo transfer and related technologies. Approximately 25,000 equine embryo transfers are being conducted per year, worldwide. [Copyright &y& Elsevier]

Abstract: The main objective was to determine the efficacy of using X-sorted sperm to produce embryos in vitro for transfer into lactating dairy cows. Cows were bred by timed artificial insemination (TAI) using nonsorted semen or X-sorted sperm, or they received a fresh embryo produced in vitro by fertilization with X-sorted or nonsorted sperm using timed embryo transfer (TET). Pregnancy rates at approximately Day 32 averaged over all dairies were 39.3 ± 3.2% (least-squares mean ± SEM) for TAI nonsorted, 27.3 ± 3.4% for TET nonsorted fresh embryos, and 30.2 ± 3.3% for TET X-sorted fresh embryos (TAI vs. both TET groups, P < 0.05; 206 to 233 cows per group). Pregnancy losses between approximately Day 32 and term ranged from 16% to 37%, the latter from TET with X-sorted sperm. Pregnancy losses to term were higher for cows receiving embryos produced in vitro than for cows bred by TAI. Calves produced via TET were not substantively different from AI controls in physical measurements or standard blood chemistry profiles. [Copyright &y& Elsevier]

Abstract: Objectives were to determine progesterone concentration (P4) from days 4 to 28 relative to presumptive estrus necessary for maintenance of pregnancy in lactating Holstein cows. Cows were assigned to the low P4 (LowP4, n =28) or control (n =153) treatments. All cows were presynchronized with two injections of PGF2α (14d apart) and an ovulation synchronization protocol (11d later; GnRH on day −10, PGF2α on day −3; and GnRH on day 0=presumptive estrus). Cows in the Low P4 treatment received 2 injections of prostaglandin F2α on days 4 and 5 (day 0=presumptive estrus) and a new CIDR insert on day 5 that was replaced every 7d until day 28. Blood was sampled on days −9, −2, 0, 4, 7,14, 21, and 28. Ovaries were examined with ultrasound on days −9, −3, and 7 and cows bearing a corpus luteum ≥20mm on day 7 received an embryo. On days 0, 4 and 7 P4 did not differ (P ≥0.27) but control cows had greater (P <0.01) P4 on days 14, 21, and 28. Progesterone concentration fold change from day 0 to 7 was not (P =0.14) affected by treatment, but P4 concentration fold change from day 7 to 14 was (P <0.01) greater for control cows compared with LowP4 cows. No LowP4 cows became pregnant after embryo transfer, whereas 35.7, 25.5, and 21.4% of control cows were pregnant on day 28, 42, and 63, respectively. Progesterone concentration fold changes from day 0 to 7 (P =0.03) and from day 7 to 14 (P =0.05) were associated with pregnancy outcomes on day 63. Among cows that were pregnant on day 63, the minimum P4 concentration fold changes from day 0 to 7 and from day 7 to 14 were 2.71 and 1.48, respectively. Interestingly, cows with P4 concentration <5ng/mL on day 14 were (P =0.01) and tended to be (P =0.07) more likely to lose pregnancy from day 28 to 42 and from day 28 and 63, respectively. Faster rise in P4 concentration during the metestrus and early diestrus are associated with pregnancy establishment following embryo transfer, which suggests that early rise in P4 concentration has an indirect effect on embryo development through modulation of uterine environment and secretion of histotroph. Furthermore, the positive effects of early rise in P4 concentration appear to go beyond the phase of maternal recognition of pregnancy through adhesion and placentation stages. [Copyright &y& Elsevier]

Abstract: In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season. [Copyright &y& Elsevier]

Abstract: Currently, timed ovulation induction and fixed-time artificial insemination (FTAI) in superstimulated donors and synchronization protocols for fixed-time embryo transfer (FTET) in recipients can be performed using GnRH or estradiol plus progesterone/progestin (P4)-releasing devices and prostaglandin F2α (PGF2α). The control of follicular wave emergence and ovulation at predetermined times, without estrus detection, has facilitated donor and recipient management. However, because Bos taurus cows have subtle differences in their reproductive physiology compared with Bos indicus cattle, one cannot assume that similar responses will be achieved. The present review will focus on the importance of orchestrating donor and recipient management to assure better logistics of procedures to achieve more desirable results with embryo collection and transfer. In addition, this will provide clear evidence that the use of FTAI in superstimulated donors and FTET in embryo recipients eliminates the need to detect estrus with satisfactory results. These self-appointed programs reduce labor and animal handling, facilitating the use of embryo transfer in beef and dairy cattle. [Copyright &y& Elsevier]

Abstract: Technologies for in vitro embryo production have the potential to enhance the efficiency of cattle production systems. However, utilization of in vitro-produced embryos for transfer remains limited throughout much of the world. Despite improvements over the past two decades, problems associated with the production of bovine embryos in vitro still exist which limit the widespread commercial application of this technology. In particular, bovine embryos produced in vitro have a reduced capacity to establish and maintain pregnancy as compared with their in vivo-derived counterparts. Embryo competence for survival following transfer is improved by in vivo culture in the sheep oviduct, thus indicating that standard embryo culture conditions are sub-optimal. Therefore, one strategy to improve post-transfer survival is to modify embryo culture media to more closely mimic the in vivo microenvironment. The maternal environment in which the bovine embryo develops in vivo contains various growth factors, cytokines, hormones, and other regulatory molecules. In addition to affecting bovine embryo development in vitro, recent research indicates that embryo competence for survival following transfer can also be improved when such molecules are added to embryo culture medium. Among the specific molecules that can increase post-transfer embryo survival are insulin-like growth factor-1 (IGF-1), colony stimulating factor-2 (CSF-2) and hyaluronan. This paper will review the effects IGF-1, CSF-2 and hyaluronan on post-culture embryo viability and discuss the potential mechanisms through which each of these molecules improves post-transfer survival. [Copyright &y& Elsevier]

Abstract: The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation. [Copyright &y& Elsevier]

Abstract: We hypothesized that administration of hCG to recipients at embryo transfer (ET) would induce accessory CL, increase serum progesterone concentrations, and reduce early embryonic loss (as measured by increased transfer pregnancy rates). At three locations, purebred and crossbred Angus, Simmental, and Hereford recipients (n = 719) were assigned alternately to receive i.m. 1,000 IU hCG or 1 mL saline (control) at ET. Fresh or frozen-thawed embryos were transferred to recipients with a palpable CL on Days 5.5 to 8.5 (median = Day 7) of the cycle (Locations 1 and 2), or on Day 7 after timed ovulation (Location 3). Pregnancy diagnoses (transrectal ultrasonography) were done 28 to 39 d (median = 35 d) and reconfirmed 58 to 77 d (median = 67 d) post-estrus. At Location 1 (n = 108), ovaries were examined at pregnancy diagnosis to enumerate CL. More (P < 0.001) pregnant hCG-treated cows (69.0%) had multiple CL than pregnant controls (0%). Serum progesterone (ng/mL) determined at Locations 1 and 2 (n = 471) at both pregnancy diagnoses in pregnant cows was greater (P ≤ 0.05) after hCG treatment than in controls (first: 8.1 ± 0.9 vs 6.1 ± 0.8; second: 8.8 ± 0.9 vs 6.6 ± 0.7), respectively. Unadjusted pregnancy rates at the first diagnosis were 61.8 and 53.9% for hCG and controls. At the second diagnosis, pregnancy rates were 58.6 and 51.3%, respectively. Treatment (P = 0.026), embryo type (P = 0.016), and BCS (P = 0.074) affected transfer pregnancy rates. Based on odds ratios, greater pregnancy rates occurred in recipients receiving hCG, a fresh embryo (66.3 vs 55.5%), and having BCS >5 (62.3 vs 55.3%). We concluded that giving hCG at ET increased incidence of accessory CL, serum progesterone in pregnant recipients, and transfer pregnancy rates. Furthermore, we inferred that increased progesterone resulting from hCG-induced ovulation reduced early embryonic losses after transfer of embryos to recipients. [Copyright &y& Elsevier]

The objective of experiment 1 was to evaluate the effects of treatments with human chorionic gonadotropin (hCG) or GnRH 7 d after induced ovulation on reproductive performance of lactating dairy cows submitted to timed artificial insemination (TAI) or timed embryo transfer (TET). A total of 834 potential breedings were used from 661 lactating Holstein cows (37.3 ± 0.3 kg of milk/d). Cows had ovulation synchronized and were assigned randomly to receive TAI on d 0 or TET on d 7. Within each group, cows were assigned randomly to receive on d 7 no additional treatment (control; nTAI = 156; nTET = 126), a 100 μg i.m. injection of GnRH (nTAI = 155; nTET = 124), or a 2,500 IU i.m. injection of hCG (nTAI = 151; nTET = 122). Postbreeding treatment affected the percentages of pregnant cows at TET on d 28 (control: 38.1%; GnRH: 52.4%; hCG: 45.1%) and on d 60 (control: 32.5%; GnRH: 41.1%; hCG: 38.5%), but postbreeding treatment did not affect percentages of pregnant cows at TAI on d 28 (control: 30.1%; GnRH: 32.2%; hCG: 32.4%) or on d 60 (control: 25.6%; GnRH: 27.1%; hCG: 29.8%). The objective of experiment 2 was to evaluate the effect of a treatment with GnRH 7 d after TET on reproductive performance of lactating dairy cows that received a previous GnRH treatment at TET. A total of 285 potential breedings were used from 257 lactating Holstein cows (35.1 ± 0.8 kg of milk/d). Cows had ovulation synchronized and were assigned for TET on d 7. Immediately after TET, all cows were treated with a 100 μg i.m. injection of GnRH. On d 14, cows were assigned randomly to receive (G7–14; n = 147) or not (G7; n = 138) an additional injection of GnRH. Pregnancy diagnosis were performed on d 28 and 60. The additional treatment with GnRH on d 14 did not affect the percentages of pregnant cows on d 28 (G7: 48.5%; G7–14: 42.9%) or on d 60 (G7: 39.8%; G7–14: 37.4%). In conclusion, treatment with GnRH or hCG 7 d after induced ovulation increased conception rates in lactating dairy cows submitted to TET, but not in cows submitted to TAI. Moreover, treatment with GnRH 7 d after TET did not enhance reproductive performance of lactating dairy cows that received a previous GnRH treatment at TET. [ABSTRACT FROM AUTHOR]

Abstract: Neosporosis is considered to be one of the main causes of abortions in bovines. In this study we evaluated the congenital transmission and occurrence of abortions by Neospora caninum in an embryo transfer center in Neropolis, Goiás. Serological samples from 101 recipients, 61 donors, and 90 calves were analyzed. Among these animals, 32.67% of the recipients, 22.22% of the donors, and 6.66% of the calves were positive for N. caninum. The rate of vertical transmission was 24%. There was a statistically significant difference between antibody titer of the recipient cows and the serological status of the calves. There was no statistically significant relationship between N. caninum serological status and reproductive rates such as estrum repetition and embryo production. Eight abortions occurred, six (75%) in positive females and two (25%) in negative females. These results indicate that neosporosis may be an important cause of failing reproduction and that in embryo transfer centers recipients should be previously examined, and those that are seronegative for Neospora should be chosen to reduce abortion and the birth of seropositive calves. [ABSTRACT FROM AUTHOR]

Abstract: The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated . In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to compare a synchronization of time of ovulation protocol for fixed-timed embryo transfer (FTET) with the usual administration of a single dose of prostaglandin associated with detection of estrus. Also, the effect of the presence of CL at the beginning of FTET protocol was evaluated. Lactating Holstein cows (n =651) with three previous artificial inseminations were classified according to presence or absence of a corpus luteum (CL). Cows with a CL were randomly assigned to two additional treatments and submitted to embryo transfer after detection of estrus (PGF-Estrus) or FTET (FTET-CL). Cows without CL were allocated to the FTET-NoCL treatment. On a random day of the estrous cycle (Day 0), cows in the PGF-Estrus treatment (n =229) were treated with 150μg d-cloprostenol (PGF) i.m. followed by detection of estrus from Day 1 through Day 5 after PGF. Embryos were transferred 6–8 days after estrus detection. Cows in the FTET-CL (n =208; presence of CL) and FTET-NoCL (n =214; absence of CL) treatments received a norgestomet ear implant plus 2mg estradiol benzoate (EB) and 50mg progesterone i.m. on Day 0. On Day 8, the implant was removed and 400IUeCG, 150μg d-cloprostenol and 1mg estradiol cypionate i.m. were administered. No detection of estrus was performed and Day 10 was arbitrarily considered as the estrus day. Ultrasonographic exams were performed in all recipients and only cows with a single CL≥15mm or multiple CL received a fresh or frozen–thawed embryo on Day 17. Pregnancy was diagnosed by ultrasonography at 30 and 60 days of pregnancy. When FTET and PGF-Estrus were compared, the proportion of cows receiving an embryo (recipients transferred-to-treated rate) was greater in the FTET-CL (75.0% (156/208) than in PGF-Estrus (34.5%, 79/229; P <0.0001) treatment. Pregnancy rate (60 days) was also greater in FTET-CL (29.3%, 61/208) when compared to PGF-Estrus (16.2%, 37/229; P =0.001). However, no differences were found in pregnancy loss [PGF-Estrus=11.9% (5/42), FTET-CL=9.0% (6/67); P =0.62] and circulating progesterone concentration at embryo transfer [PGF-Estrus=4.02±0.52ng/mL (n =25), FTET-CL=3.33±0.32ng/mL (n =27); P =0.25] among these treatments. The presence of CL at the beginning of FTET protocol resulted greater transferred-to-treated rate [FTET-CL=75.0% (156/208) vs. FTET-NoCL=61.2% (131/214); P =0.003], but showed no effect on pregnancy rate at 60 days [FTET-CL=29.3% (61/208) vs. FTET-NoCL=22.9% (49/214); P =0.13], pregnancy loss [FTET-CL=9.0% (6/67) vs. FTET-NoCL=2.0% (1/50); P =0.15] and circulating progesterone concentration at ET [FTET-CL=3.33±0.32ng/mL (n =27) compared to FTET-NoCL=3.44±0.40ng/mL (n =2 9); P =0.82]. In conclusion, the protocol for synchronization of time of ovulation using norgestomet ear implant, EB and eCG increased recipients transferred-to-treated and pregnancy rates in high-producing repeat-breeder Holstein cows. Also, recipients without CL at the beginning of the time of ovulation synchronization treatment resulted in similar pregnancy rate as recipients with CL submitted to FTET protocol. Thus, the suggested protocol allowed the performance of FTET, without the need for detection of estrus, simplifying the reproductive management and increasing the reproductive efficiency in repeat-breeder Holstein recipients. [Copyright &y& Elsevier]

Objective: To evaluate the effect of the biopsy of 8-cell to 16-cell bovine embryos on their subsequent development and the effect of whole genome amplification (WGA) on removed blastomeres. Design: Randomized study. Setting: Molecular genetics and animal reproduction laboratories. Patient(s): Cow ovaries obtained from slaughterhouses. Intervention(s): The ovaries were punctured, and the oocytes were matured and fertilized in vitro. On the fourth day after fertilization, 8-cell to 16-cell bovine embryos were biopsied, one quarter of each embryo being removed. The blastomeres were submitted to WGA followed by polymerase chain reaction (PCR). The embryos were returned to culture for evaluation of their development. Main Outcome Measure(s): Subsequent rate of blastocyst development, embryo cell number, WGA efficiency, and sex determination. Result(s): A total of 92 embryos were submitted to biopsy. The blastocyst production was 53.3%, with 44.9% of hatching rate. These results were similar to those of the control group (66.0% and 42.6%) of 103 embryos. Overall, no impact was detected on embryo quality in blastocyst cell number between the two groups. Removed blastomeres were submitted to WGA, resulting in 98.2% of efficiency. However, only 59% of the samples were sexed by PCR. Conclusion(s): Biopsy of 8-cell to 16-cell bovine embryos did not affect their subsequent development. WGA was successful in removed blastomeres. [Copyright &y& Elsevier]

Abstract: The objective was to investigate the influence of corpora lutea physical and functional characteristics on pregnancy rates in bovine recipients synchronized for fixed-time embryo transfer (FTET). Crossbred (Bos taurus taurus×Bos taurus indicus) nonlactating cows and heifers (n=259) were treated with the following protocol: 2mg estradiol benzoate (EB) plus an intravaginal progesterone device (CIDR 1.9g progesterone; Day 0); 400 IU equine chorionic gonadotropin (eCG; Day 5); prostaglandin F2α (PGF2α) and CIDR withdrawal (Day 8); and 1mg EB (Day 9). Ovarian ultrasonography and blood sample collections were performed on Day 17. Of the 259 cattle initially treated, 197 (76.1%) were suitable recipients; they received a single, fresh, quality grade 1 or 2 in vivo–derived (n=90) or in vitro–produced (n=87) embryo on Day 17. Pregnancy rates (23 d after embryo transfer) were higher for in vivo–derived embryos than for in vitro–produced embryos (58.8% vs. 31.0%, respectively; P<0.001). Mean (±SD) plasma progesterone (P4) concentration was higher in cattle that became pregnant than that in nonpregnant cattle (5.2±5.0 vs. 3.8±2.4ng/mL; P=0.02). Mean pixel values (71.8±1.3 vs. 71.2±1.1) and pixel heterogeneity (14.8±0.3 vs. 14.5±0.5) were similar between pregnant and nonpregnant recipients (P>0.10). No significant relationship was detected between pregnancy outcome and plasma P4, corpus luteum area, or corpus luteum echotexture. Embryo type, however, affected the odds of pregnancy. In conclusion, corpus luteum–related traits were poor predictors of pregnancy in recipients. The type of embryo, however, was a major factor affecting pregnancy outcome. [Copyright &y& Elsevier]

Abstract: We tested the hypothesis that subclinical endometritis occurs after embryo transfer (ET) in the horse. Recipient mares were treated with meclofenamic acid (M) or flunixin meglumin (F) after ET or were left untreated (n =9 per group). Embryos were re-collected 4 days after transfer. Endometrial biopsies were taken for histology and analysis of cyclooxygenase-2 (COX-2) by immunohistochemistry and for PCR. Bacteriological swabs were collected from the uterus and lavage fluid of donor and recipient mares. Progesterone and prostaglandin F2α release was analysed in recipient mares after ET. Four days after ET, four embryos were recovered from group M and three from group F and untreated mares, each. The number of polymorph nuclear neutrophils was reduced in treated mares (p <0.05). Expression of mRNA for inflammatory cytokines did not differ between groups. In group M, expression of endometrial prostaglandin-E-synthase was higher than in group F (p <0.05). Three out of nine control mares unterwent preterm luteolysis (p <0.05 vs. treatment groups), prostaglandin release (p <0.05) and the number of COX-2 positive cells (p <0.01) were significantly higher than in treated mares. Only few bacteriological swabs were positive. In conclusion, treatment of embryo recipient mares with non-steroid anti-inflammatory drugs inhibits the inflammatory response of the endometrium after ET. Meclofenamic acid may have advantages in comparison to flunixin meglumin due to a different influence on prostaglandin synthesis that may not result in inhibition of embryonic mobility. [Copyright &y& Elsevier]

Abstract: The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation. [Copyright &y& Elsevier]

Abstract: The timing between round spermatid(s) (RS) injection and oocyte activation are critical for spermatid remodeling and embryo development in intracytoplasmic injection of round spermatid (ROSI) procedure. The objective of the present study was to develop an appropriate oocyte activation method for producing developmentally competent bovine embryos reconstructed with RS. Embryos reconstructed by ROSI were compared with three activation treatments for the rates of pronuclear formation, development and ploidy. RS were isolated from bull testes by Percoll density gradients. Matured oocytes were divided into three activation groups. In Group 1, oocytes were activated with ionomycin (5μM, 5min) before ROSI. In Group 2, oocytes were activated with ionomycin after ROSI. In Group 3, oocytes were activated twice with ionomycin before and after ROSI. All the eggs were then incubated in cycloheximide (CHX, 10μg/mL) for 5h and cultured in CR1aa medium for up to 8 days. Three methods of oocyte activation were also compared for the activation and development of parthenotes. Activation rates among the groups were 70–79% and did not differ. Cleavage rates in parthenotes were significantly (P <0.05) higher in Group 3 than in Groups 1 and 2, but blastocyst rates did not differ among the groups. In ROSI embryos, the rates of cleavage and development into blastocysts were significantly (P <0.05) greater in Group 3 (82.3% and 13.1%) than in Groups 1 and 2 (53.7, 5.8% and 64.2, 1.7%, respectively). Ploidy analysis by examining the metaphase spreads of ROSI blastocysts displayed greater numbers of diploid chromosomal complements. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation followed by CHX treatment is more efficient for producing developmentally competent embryos. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Over the past 20 years the rate of blastocyst development in vitro has improved through the development of sequential defined media, refining the oxygen concentrations during culture and providing substrates to ameliorate free radical accumulation. Despite these advances there has been little progress in improving calving rates after the transfer of in vitro produced embryos. This suggests that the culture conditions have been very effective in enabling those fertilised oocytes to reach the blastocyst stage that otherwise would not occur in vivo.We suggest that the next advance by which the embryo transfer technology gains more acceptance in cattle production will be identifying those cows which are intrinsically superior recipients. This must be coupled to the development of non-invasive assessments of the developmental competence of both the oocyte and the blastocyst. Until these two goals are achieved the ET industry will remain static and unable to overcome the economic loss caused by embryo mortality occurring 7–10 days after transfer. [Copyright &y& Elsevier]

The exploitation of the domestic animals species of South American camelids is of great social importance for the native people living in the High Andes. The reproductive physiology of these species is a unique challenge in the development of advanced breeding techniques. At present, the cryopreservation of embryos has not been developed and very few investigations have been conducted. The objective of the present work was to evaluate the in vivo survival of vitrified llama embryos after transfer to recipient females. Donors females were treated with a CIDR-estradiol benzoate-eCG regimen and were mated naturally 6 days after CIDR withdrawal. One ovulatory dose (8 μg) of GnRH was administered immediately after mating. A second mating was allowed 24 h later. Embryo recovery was performed nonsurgically between 8 and 8.5 days after the first mating. Twenty-two ova/embryos were recovered from 12 donor females. Hatched blastocysts were exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 M sucrose + 0.375 M glucose + 3% polyethylene glycol (P/V)) in three steps, and after loading into 0.25 ml straws, were plunged into liquid nitrogen. For embryo transfer, recipients animals were ovulation-synchronized using GnRH administered at the same time as donors. A total of eight vitrified-warmed embryos and 12 fresh embryos were nonsurgically transferred to four and six recipient females, respectively (two embryo per recipient). The pregnancy rates were 50 and 33.3% for recipients that had received vitrified embryos and fresh embryos, respectively. The results demonstrated the effectiveness of this simple vitrification method for cryopreservation of llama embryos. [Copyright &y& Elsevier]

The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol+glycerol (Eg+Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg+Gly+ 0.1 M sucrose group had a greater rate as compared with Eg+Gly+0 M sucrose and Eg+Gly+0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6+Day 7 embryos; and, Eg+Gly+0.3 M sucrose group had a greater rate as compared with the Eg+Gly+0 M sucrose and Eg+Gly+0.5 M sucrose groups in evaluations of Day 6 and Day 6+Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg+Gly+0.1 M and Eg+Gly+0 M sucrose groups and the Eg+Gly+0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg+Gly+0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs. [Copyright &y& Elsevier]