Characterisation of pregnancy losses after embryo transfer by measuring plasma progesterone and bovine pregnancy-associated glycoprotein-1 concentrations.

The aim of this analysis was to determine whether pregnancy loss (PL) after embryo transfer (ET) in cattle was related to maternal progesterone (P4) concentrations during and shortly after ET, and maternal bovine pregnancy-associated glycoprotein-1 (bPAG-1) concentrations in plasma at days 25-35 of gestation. Embryos (n=260) were produced either in vivo after superovulation (n=115), or in vitro from oocytes (obtained with ovum pick-up) in co-culture (n=44) or cultured in a synthetic medium (n=101). Overall, PL was 56.9% (148) and no significant differences occurred in calving rate among the three embryo production groups. There was no difference in P4 concentrations on days 7-14 of gestation in the three groups, nor between ongoing and interrupted pregnancies. Between days 25 and 35 of pregnancy, bPAG-1 concentrations were unaffected by embryo production, but in cattle that had PL between days 26 and 120, four bPAG-1 profiles could be detected. Between days 25 and 32, bPAG-1 concentrations were influenced by PL, and concentrations were significantly lower in animals in which PL occurred between days 26 and 120 than in those animals that aborted later or calved at term. Early P4 concentrations suggested that maternal luteal factors were not responsible for PL which appeared to be caused by impaired conceptus development (regardless of embryo type) as reflected by low maternal bPAG-1 concentrations prior to embryonic death.

Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos.

Many environmental conditions for in vitro embryo production (IVP) systems for cattle have been relatively standardised, e.g. media composition, temperature, pH, water quality, and atmospheric composition. However, little attention has been paid to the quality of ambient laboratory air and the gas environment in incubators. Although a few studies have examined the effects of chemical air contamination on IVP of human embryos, there are no published accounts for domestic animal embryos. Therefore, this study investigated the effects of an intra-incubator carbon-activated air filtration system (CODA) during in vitro culture (IVC) on embryonic development and subsequent pregnancy rate of bovine embryos. Immature cumulus-oocyte-complexes (COCs) were obtained twice-weekly by ultrasonic-guided transvaginal oocyte aspiration. The COCs were matured in TCM199/FCS/LH/FSH, fertilized with frozen-thawed Percoll-separated semen, and subsequently cultured for 7 day in SOFaaBSA. Day 7 embryos were transferred either fresh or frozen/thawed. The experimental design was a 2 x 2 factorial; presumptive zygotes were placed either in a conventional CO(2)-O(2)-N(2) incubator (Control group) or in an identical CO(2)-O(2)-N(2) incubator with a CODA intra-incubator air purification unit (CODA group) for IVC. The embryo production rate at Day 7 was not affected by the CODA air purification unit (23.4 and 24.7% morulae and blastocysts per oocyte for control and CODA, respectively) nor was there any significant effect on embryo stage or quality. However, the pregnancy rate was improved (P=0.043) for both fresh (46.3% versus 41.0%) and frozen/thawed embryos (40.8% versus 35.6%). In conclusion, atmospheric purification by the CODA intra-incubator air purification unit significantly increased pregnancy rate following transfer of in vitro-produced bovine embryos.

Ways to improve the biosecurity of bovine semen.

Semen production and trade is a worldwide industry. A framework, based on international standards is awaiting international and national regulation. In the perspective of biosecurity of the final product, critical notes can be made according to the semen production regulation and product safety. Process description brings the obligatory health standards for the production bulls, collection and processing of semen, identification, registration, worldwide distribution and insemination into discussion. Test frequency, test quality and demands, way of sampling and test consistency can influence product safety. New scientific knowledge can influence the value of the regulation. Whether a country is free of notifiable disease should influence decisions regarding necessary tests for the production bulls. The biosecurity of the semen production process is influenced by several factors. The effectiveness of the antibiotics used is questionable. The extenders for cryopreservation added to the semen can affect product safety. The way materials and storage containers have to be disinfected must be clear. In modern industry, tracking and tracing is an important issue. Worldwide differences in ways of identification of straws do not benefit a proper identification and registration throughout the process. Regulation could help improve the transparency of production and trade. Before anything concerning biohazard is implemented in regulation, each rule should be thoroughly based on scientific research where possible and furthermore it must be possible to enforce the regulation. The effort it takes to enforce the regulation should be in balance with the benefit it provides. An approach to alter regulation quickly is advisable. To produce a safe product that is accepted for international trade is of vital interest for the survival of artificial insemination (AI) in cattle.

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

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

Factors affecting oocyte quality and quantity in commercial application of embryo technologies in the cattle breeding industry.

With the introduction of multiple ovulation, embryo recovery and transfer techniques (MOET) plus embryo freeze-thaw methods in the early 1980s, the breeding industry has the tools in hand to increase the number of calves from donors of high genetic merit. In the early 1990s, the introduction of ovum pick-up followed by in vitro embryo production (OPU-IVP) opened up even greater possibilities. Using these technologies, we challenge biological mechanisms in reproduction. Where normally one oocyte per estrous cycle will develop to ovulation, now numerous other oocytes that otherwise would have degenerated are expected to develop into an embryo. Completion of oocyte growth and pre-maturation in vivo before final maturation both appear to be essential phases in order to obtain competence to develop into an embryo and finally a healthy offspring. In order to increase oocyte quality and quantity in embryo production technologies, current procedures focus primarily on improving the homogeneity of the population of oocytes with regard to growth and state of pre-maturation at the start of a treatment. In the case of MOET, dominant follicle removal (DFR) before superovulation treatment improves the number of viable embryos per session from 3.9 to 5.4 in cows but not in heifers and a prolonged period of follicle development obtained by preventing release of the endogenous LH surge increases the number of ova but not the number of viable embryos per session. In the case of OPU-IVP, the frequency of OPU clearly affects quantity and quality of the collected oocytes and FSH stimulation prior to OPU every 2 weeks resulted in 3.3 embryos per session. Analysis of 7,800 OPU sessions demonstrated that the oocyte yield is dependent on the team, in particular, the technician manipulating the ovaries. It is concluded that an increased understanding of the processes of oocyte growth, pre- and final maturation will help to improve the efficiency of embryo technologies. However, somewhere we will meet the limits dictated by nature.

Effects of different reproduction techniques: AI MOET or IVP, on health and welfare of bovine offspring.

Since the introduction of in vitro production (IVP) of bovine and sheep pre-implantation embryos, increased birth weights and other deviations of IVP calves and lambs compared with AI or MOET offspring have been reported. Study 1 of the present paper, a comparison between AI, MOET and IVP (co-culture/serum) calves with respect to calf and calving characteristics in large-scale field conditions, confirms these reports. In addition, it is shown that MOET calves tend towards higher birth weights and have significantly longer gestations and more difficult calvings than AI calves. It is presently unknown if the effect of IVP is also observed later in life. In this paper, data on reproduction characteristics of bovine IVP co-culture/serum offspring are presented. Semen production--and non return data of one year old IVP bulls and superovulation-, AI- and OPU/IVP results of one year old IVP heifers are compared with those of one year old AI and MOET animals producing semen or embryos in the same time period. So far, there are no indications that the use of IVP is reflected in deviate reproduction characteristics of bovine IVP offspring. It has been suggested that use of co-culture cells and serum during in vitro culture of bovine (and sheep) embryos may partially explain the increased birth weights and other deviations of bovine and sheep IVP offspring. Deletion of these factors in semi-defined culture media, e.g. Synthetic Oviductal Fluid (SOF), could result in more normal offspring. Study 2 investigates this hypothesis in both field conditions (Study 2a, comparing AI, IVP co-culture/serum and IVP SOF calves) and in semi-standardized conditions (Study 2b, comparing MOET, IVP co-culture/serum and IVP SOF calves at one herd). In Study 2a, although IVP SOF calves showed (non-significant) shorter gestations, easier calvings and lower percentages of perinatal mortality and congenital malformations than IVP co-culture calves, birth weights were not decreased. In Study 2b however, the difference between IVP co-culture and IVP SOF calves in birth weight and ease of calving was significant (P < 0.05), IVP SOF calves resembling MOET calves more. IVP calves differed significantly from MOET calves with respect to several physiological parameters, such as blood oxygen saturation level, heart beat frequency and some measures of the heart. In addition, in Study 2b, recipients receiving an IVP SOF embryo showed a more regular return to estrus than those receiving an IVP co-culture embryo. From Study 2 it can be concluded that using a semi-defined medium for in vitro culture (SOF) may improve characteristics of IVP calves born.