Effect of cumulus cells and sperm concentration on fertilization and development of pig oocytes.

Effect of sperm concentrations and cumulus cells (CCs) on porcine IVF was re-evaluated using current improved IVM and IVC system. Our results showed that both CCs and sperm concentration had significant effect on penetration rate, frequency of polyspermy and embryonic development. The best IVF results were obtained with oocytes with CCs fertilized with 0.5 × 105 sperm/ml. Such an IVP system works on both sow and gilt oocytes.

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

Milk yield and estrous behavior during eight consecutive estruses in Holstein cows fed standardized or high energy diets and grouped according to live weight changes in early lactation.

Cows managed for extended lactation go through several estruses before rebreeding. The aims of this study were (1) to quantify the effect of the first 8 estruses after calving on milk yield, milking frequency, and estrous behavioral activity, and (2) to determine the effects of early lactation live weight gain (LWG) as an indication of energy balance on milk yield, plasma insulin-like growth factor 1 (IGF-1) concentration, estrous behavioral activity, interval from calving to first estrus, between-estrus intervals, and pregnancy risk. Milk yield, live weight, and estrous behavioral activity were measured daily in 62 Holstein cows, 17 primiparous and 45 multiparous, managed for an 18-mo calving interval. Blood plasma obtained at wk 3, 5, 12, and 24 after calving was analyzed for IGF-1. Estrus was detected by use of milk progesterone profiles combined with visual observations (i.e., mounting behavior and other). The cows were divided into 2 groups: the cows having a negative LWG in each of the first 5 wk postpartum and the cows having a positive LWG in at least 1 of the first 5 wk after calving. The results indicate a similar decrease of 0.56 kg of milk per day of estrus during each of the 8 consecutive estruses. The activity level was 17 ± 1 movements per hour higher during the 8 estruses compared with the basic activity level. More cows expressed mounting behavior at estrus 8 than at estrus 2 (63.3 and 45.9%, respectively). The negative LWG cows had lower IGF-1 and higher milk production than the positive LWG cows. Both LWG groups had similar interval from calving to first estrus, on average 55 d. To conclude, the decrease in milk yield during estrus is marginal and similar in consecutive estruses. Moreover, estrous behavior is more highly expressed in the later estruses compared with the earlier estruses. Reproductive parameters (frequency of mounting, pregnancy risk, interval to first estrus, and between-estrus intervals) were not influenced by the live weight change during early lactation.

Postmortem findings in cloned and transgenic piglets dead before weaning.

Important factors contributing to the well-known high mortality of piglets produced by SCNT are gross malformations of vital organs. The aim of the present retrospective study was to describe malformations found in cloned piglets, transgenic or not, dying or culled before weaning on Day 28. Large White (LW) embryos were transferred to 78 LW recipients, while 72 recipients received Göttingen embryos (67 transgenic and five not transgenic) and 56 received Yucatan embryos (43 transgenic and 13 not transgenic). Overall pregnancy rate was 76%, and there were more abortions in recipients with minipig embryos than in those with LW embryos (26% and 24% vs. 6%). Piglets (n = 815) were born from 128 sows with 6.5 ± 0.4 full-born piglets per litter. The overall rate of stillborn piglets was 21% of all born with the number of stillborn piglets ranging from one to nine in a litter. The mortality of the surviving piglets during the first month was 48%. Thus, altogether 58% of the full-born piglets died before weaning. In 87 of the 128 litters (68%), one to 12 of the piglets showed major or minor malformations. Malformations were found in 232 piglets (29.5% of all born). A single malformation was registered in 152 piglets, but several piglets showed two (n = 58) or more (n = 23) malformations (7.4% and 2.8% of all born, respectively). A significantly higher malformation rate was found in transgenic Göttingen and Yucatan piglets (32% and 46% of all born, respectively) than in nontransgenic LW (17%). There was a gender difference in the transgenic minipigs because male piglets had a higher rate of malformations (49.1%) than females (29.7%). The most common defects in the cloned piglets were in the digestive (12.2%), circulatory (9.4%), reproductive (11.3%), and musculoskeletal (9.1%) systems. Malformations of the musculoskeletal system were most frequent in Göttingen (16.3% vs. approximately 5.5% in the two other breeds), whereas abnormal cardiopulmonary systems were most frequent in Yucatan piglets (26.9% vs. 2.1% in LW and 5.3% in Göttingen). In conclusion, these results show that pig cloning results in a considerable loss of piglets and that many of these can be related to various malformations that all are also seen in noncloned piglets. Because approximately half of the cloned piglets still survive, even with eventual unknown minor malformations, use of pigs as models for human diseases is still realistic. However, continued efforts are needed to further reduce the level of malformations.

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer.

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter ('small' ≤110µm; 'medium' >110µm; 'large' ≥120µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

Developmental kinetics of pig embryos by parthenogenetic activation or by handmade cloning.

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed.

Uterine bacterial flora in postpartum Danish Holstein dairy cows determined using DNA-based fingerprinting: correlation to uterine condition and calving management.

The overall aim of this study was to describe uterine bacterial flora during the postpartum period in Danish Holstein cows using the Terminal Restriction Fragment Length Polymorphism (T-RFLP) method. This method produces a pattern of nucleic acid fragments from the microorganisms present, reflecting the "fingerprint" of the actual microbial flora. As well as characterizing changes in flora with time from calving and between herds, data were examined for strong relations between uterine bacterial flora, calving management and uterine condition. In total 125 Holstein cows from five herds were included, and for each cow calving management was recorded. Cows were clinically examined on average 8 (range 0-19) and 28 (range 22-38) days after calving, and a uterine sample was taken for bacterial identification using T-RFLP. Milk samples were taken weekly for progesterone analysis. Bacteria were found in all cows at both examinations, and the flora was composed of many species, including species not traditionally reported to be present in the bovine uterus. The bacterial composition differed according to days from calving and herd. In all five herds Fusobacterium necrophorum, Pseudomonas/Acinetobacter and Bacteroides/Sphingobacterium/Prevotellaceae were among the most common at both examinations. In four herds there was a percentage decrease of F. necrophorum from first to second examination, and in all herds there was a percentage increase of Pseudomonas/Acinetobacter from first to second examination. No differences in bacterial flora were found between cows with different uterine scores, which were influenced by herd, calving difficulty and retained placenta.

Short communication: Large-scale study on effects of metritis on reproduction in Danish Holstein cows.

A total of 398,237 lactations of Danish Holstein dairy cows were studied with the main objective to investigate the effects of metritis on 2 fertility variables: interval from calving to first insemination (CFI) and nonreturn rate at 56 d after first insemination (NR56), adjusting for milk production and body condition score as confounders. Metritis was defined as a score of at least 5 (indicating purulent vaginal discharge with abnormal smell) on the Danish uterine score scale (from 0 to 9, used to evaluate vaginal discharge in the first 19 d postpartum on all fresh cows in herds participating in a national herd health program). Cows with metritis in early lactation presented a significant delay in first insemination (hazard ratio of 0.80) and a significantly reduced probability of success at first insemination. The effect of metritis was also present after adjusting for possible effects of body condition score, milk production in the first month of lactation, parity, herd, and year-season. Only a small part of the observed effect of metritis could be explained by variation in body condition score at calving and milk production in the first month after calving. The results from this large-scale study underscore the deleterious effects of metritis on the fertility variables CFI and NR56.

Establishment of an efficient somatic cell nuclear transfer system for production of transgenic pigs.

Handmade cloning (HMC) is now an established procedure used in several species for somatic cell nuclear transfer, but only applied in two related laboratories for pigs. The aim of this review is to facilitate widespread application by summarizing the process of establishment and explaining the background of the incorporated special approaches. Optimized steps of traditional cloning in pigs (in vitro maturation, activation, embryo culture) were merged with those of the micromanipulation-free HMC that has been modified according to the specific needs of sensitive porcine oocytes (partial zona digestion before enucleation, two-step zona-free fusion with the somatic cell; initiation of activation with the second fusion). The zona-free approach required embryo culture to the blastocyst stage before surgical transfer of embryos to the uterine horns of recipient sows in the proper phase of an unstimulated cycle. Eventually a competitive, inexpensive and reliable alternative to traditional porcine nuclear transfer cloning techniques evolved that is also suitable to produce transgenic offspring containing various genetic modifications to establish models for several human diseases with genetic background. Further improvements and involvement of additional techniques to increase the overall efficiency and facilitate practical applications are expected in the foreseeable future.

Variation in macrophage migration inhibitory factor [MIF] immunoreactivity during bovine gestation.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in several aspects of the immune response. MIF appears to play important roles in materno-fetal immuno-tolerance during placental establishment, modulation and growth as studied in epitheliochorial porcine and hemochorial human and mouse placentae. Here we studied the bovine placenta being multiplex, villous and synepitheliochorial with a low degree of invasion, to see if MIF could be involved. Placental tissues sampled from 12 cows at 9 stages of gestation (days 18-250), and endometrial tissues from two non-pregnant animals were processed for immunohistochemistry. Bovine MIF was detected by Western blot using anti-human MIF monoclonal antibodies. An immunoreactive band of approximately 12kDa confirmed similarities between bovine and human MIFs. Compared to the non-pregnant stage with very faint staining, the caruncular epithelium during pregnancy showed stronger staining for MIF. The intercaruncular epithelium in non-pregnant endometrium showed some reaction apically with increasing intensity at uterine gland openings; in contrast, at day 18 of gestation this staining was markedly increased. During gestation both caruncular and trophoblast epithelium of the placentomes were positive with different intensity in relation to the gestational stage. In the uterine glands, some strongly stained cells were present. The mature binucleated trophoblast giant cells were negative throughout pregnancy. During reestablishment of vascularisation, the vasculature in the caruncular area showed MIF reactivity. While supporting involvement of MIF in different placental types, the spatio-temporal variation in the bovine placenta suggests a regulatory role for MIF mainly in the interhemal barrier and during vascular development.

Maternal endometrial oedema may increase perinatal mortality of cloned and transgenic piglets.

The perinatal mortality of cloned animals is a well-known problem. In the present retrospective study, we report on mortality of cloned transgenic or non-transgenic piglets produced as part of several investigations. Large White (LW) sows (n = 105) received hand-made cloned LW or minipig blastocysts and delivered either spontaneously or after prostaglandin induction followed by either Caesarean section or vaginal birth. The overall pregnancy rate was 62%, with 26% of pregnancies terminating before term. This resulted in 48 deliveries. The terminated pregnancies consisted of 12 abortions that occurred at 35 ± 2 days gestation and five sows that went to term without returning to heat and then by surgery showed the uterus without fetal content. The gestation length was for sows with LW piglets that delivered by Caesarean section or vaginally was 115.7 ± 0.3 and 117.6 ± 0.4 days, respectively. In sows with minipiglets, the gestation length for those delivered by Caesarean section or vaginally 114.4 ± 0.2 and 115.5 ± 0.3 days, respectively. Of the 34 sows that delivered vaginally, 28 gave birth after induction, whereas 6 farrowed spontaneously. Of the 14 sows that delivered after Caesarean section and in the five empty sows, the endometrium and placenta showed severe oedema. Piglet mortality following vaginal delivery was higher than after Caesarean section (31% v. 10%, respectively; P < 0.001). When vaginal delivery occurred spontaneously, the stillborn rate was greater than after induced delivery (56% v. 24%, respectively; P < 0.0001). Internal organ weights were recorded for seven cloned LW piglets and six normal piglets. The relative weight of the heart, liver, kidneys and small intestine was found to be reduced in the cloned piglets (P < 0.05). The present study demonstrates extensive endometrial oedema in sows pregnant with cloned and transgenic piglets, as well as in empty recipients, at term. The growth of certain organs in some of the cloned piglets was reduced and the rate of stillborn piglets was greater in cloned and transgenic piglets delivered vaginally, possibly because of oedema of the fetal-maternal interface.

A universal index of uterine discharge symptoms from calving to 6 weeks postpartum.

In this paper, a new uterine discharge index (D-index) was created and tested. It was based on a principal component analysis (PCA) of clinical findings of classical uterine discharge symptoms and rectal temperature during the postpartum period of dairy cattle. The PCA analysis revealed how uterine discharge features relate to each other and how they cluster together possibly representing different degrees of uterine inflammation. The D-index was the result of the multivariate PCA-analysis, and the D-index gives a continuous value between 0 and 10. It was demonstrated that the same scale, i.e. the D-index, can be used without any adjustment from 1 to 6 weeks post-calving. It is valid for any type of uterine discharge without defining the type of infection or differentiating between infection and contamination. The D-index was tested using the uterine involution data. Uterine involution was significantly delayed in the high-D-index group of cows. Similarly, in the test with all cows, involution was progressively delayed and the rate of involution of the pregnant horn was slowed down with the increase of the D-index values. It is concluded that the D-index can be a new practical, universal, tool for improved management of dairy cows in the postpartum period under commercial conditions.

Pregnancies and piglets from large white sow recipients after two transfer methods of cloned and transgenic embryos of different pig breeds.

The aim of this study was to report from a larger study with pregnancy and delivery results after transfer of cloned transgenic/non-transgenic Large White or minipig embryos to Large White sow recipients. The effect of both total numbers of transferred embryos as well as site of their deposition (uni- vs. bi-lateral) was studied. Four to five days after natural heat, 85 Large White (LW) sows received Day 5 or 6 handmade cloned embryos. Large White embryos were non-transgenic and were transferred to 36 recipients, while 49 recipients each received Minipig embryos, either non-transgenic or with 1 of 4 types of transgenes. Furthermore, the number of embryos transferred was in two categories, as 46 recipients received 40-60 embryos while 39 received 60-120 embryos. Finally, in 59 of the recipients embryos were transferred to one of the uterine horns (unicornual) while 26 other recipients had embryos transferred to both uterine horns (bicornual). The overall pregnancy rate was 55% with an abortion rate of 26% resulting in 41% deliveries with no difference between LW and Minipig embryos and no difference between transgenic and non-transgenic Minipig embryos. Transfer of 60-120 embryos resulted in more pregnancies and deliveries (62%) than <60 embryos (24%). The mean litter size was 5.1 ± 0.5 and after transfer of 60-120 embryos significantly higher (6.0 ± 0.5) than after transfer of <60 embryos (3.5 ± 0.8). Also, the bicornual transfer resulted in significantly higher delivery rate (74% vs. 44%) and mean litter size (6.1 ± 0.7 vs. 4.2 ± 0.6) than the unicornual. The mean rate of piglets/transferred embryos was 7.3 ± 0.6% while the mean rate of piglets/reconstructed embryos was 179/18,000 = 1% with no difference between breeds or number of embryos transferred. The overall perinatal mortality rate was 49%, and it was significantly lower in LW piglets (20/59 = 34%) than in Minipiglets (67/120 = 56%) (vs. 10-15% in normal piglets at the farm) and the total rate of piglets with one or more malformation was 22%, and lower in LW (12%) than in Minipiglets (28%). This study demonstrate that although the perinatal mortality was rather high, an acceptable birth rate can be achieved after transfer to LW recipients of cloned LW embryos as well as cloned, transgenic/non-transgenic Minipig embryos. Furthermore, the pregnancy rate and litter size were correlated to the number of embryos transferred and to bicornual transfer.

Respiration rates correlate with mRNA expression of G6PD and GLUT1 genes in individual bovine in vitro-produced blastocysts.

Quantification of embryo respiration is a promising procedure to assess embryonic metabolism and possibly select viable embryos. At the blastocyst stage, ATP is produced by glycolysis and oxidative phosphorylation, processes that require uptake of oxygen and glucose, which is regulated by the expression of GLUT1 and G6PD. The purpose of the present study was to investigate the relationship between respiration rates and relative abundances of G6PD and GLUT1 transcripts in individual bovine blastocysts produced in vitro. Respiration rates of 104 bovine in vitro-produced blastocysts were measured individually using the nanorespirometer technology. Real-time RT-PCR was employed to determine the relative abundance of G6PD and GLUT1 mRNA in individual embryos. The mean respiration rates were similar for male and female blastocysts of the same developmental stage, but the sex ratio was skewed towards males. GLUT1 expression was down-regulated in female versus male embryos. In contrast, a approximately 1.8-fold increase in the expression of G6PD mRNA was observed in female blastocysts when compared to male blastocysts, indicating that dosage compensation for this gene had not yet occurred. Both GLUT1 and G6PD expression levels were affected by morphological quality and stage of development. Expression of GLUT1 and G6PD mRNAs was correlated with respiration rates, indicating that, in metabolically active blastocysts, uptake of oxygen and glucose are jointly increased. These findings suggest that expression of genes for oxidative phosphorylation and glycolysis are both involved in oxygen demanding ATP production.

Investigation of respiration of individual bovine embryos produced in vivo and in vitro and correlation with viability following transfer.

BACKGROUND: Quantification of oxygen consumption by individual preimplantation embryos has the potential to improve embryo selection. This study investigated whether respiration rates of individual embryos are useful indicators of embryo viability. The effect of the Nanorespirometer on embryo viability was also evaluated. METHODS: The respiration rates of individual day 7 bovine in vivo- (n=44) and in vitro-produced (n=156) embryos were measured using the Nanorespirometer. In vivo-produced embryos were individually transferred to recipients. RESULTS: The respiration rates of in vivo-produced embryos increased with increasing morphological quality and stage of development (P < 0.05). Pregnancy rates on days 35 and 60 were 65 and 60%, respectively. The mean respiration rate did not differ significantly between embryos producing and not producing a pregnancy, but the transfer of embryos with respiration rates <0.78 nl/h, between 0.78 and 1.10 nl/h, and >1.10 nl/h resulted in 48, 100 and 25% pregnancy rate, respectively. The mean respiration rate of in vitro-produced embryos was higher than that of in vivo-produced embryos because of differences in the morphological quality and stage of development. CONCLUSION: The Nanorespirometer does not adversely influence embryo viability, but the sample size was too small to confirm the significance of the correlation observed between respiration rates and viability.

Effect of days post-partum, breed and ovum pick-up scheme on bovine oocyte recovery and embryo development.

The objective of this study was to investigate (i) the effect of two different ovum pick-up (OPU) schemes (once vs twice weekly aspirations) on oocyte recovery rate, quality and subsequent in vitro embryo development, (ii) the influence of days post-partum on oocyte recovery and (iii) possible differences in OPU results from two different herds. In group A, OPU was performed twice weekly in two Holstein Friesian (HF) and three Danish Red and White (DRW) cows from a private herd. In the research herd, two groups of eight HF cows were investigated: group B (OPU once weekly) and group C (OPU twice weekly). The collected oocytes were subsequently submitted to in vitro embryo production. More oocytes were recovered from the private herd when compared with the research herd. In the research herd, the twice weekly scheme aspirated more oocytes than the once weekly scheme. The quality of the retrieved oocytes was significantly different between groups B and C but not between groups A and C, and HF cows yielded higher quality oocytes than DRW cows (p = 0.029). Oocytes from group C showed higher level of embryonic development than group B oocytes. No differences in blastocyst rates were observed between groups A and C. Session affected the number of retrieved oocytes and subsequent developmental rates, with these being lower in the first compared with the last sessions. Finally, there was no significant effect of days post-partum in the number and quality of the retrieved oocytes, likely because of the small group size and high variation between sessions.

Localization of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in bovine placentomes from implantation until term.

Interactions of vascular endothelial growth factor (VEGF) with its receptors VEGFR-1 and VEGFR-2 promoting angiogenesis have been described in placentation of human, mink and pig. The bovine placenta is multiplex, villous and synepitheliochorial due to migratory trophoblast giant cells (TGC). To determine the role of VEGF in bovine implantation and placentation, placentomes and interplacentomal areas from 33 cows from early implantation until near term were evaluated by immunohistochemistry. VEGF immunoreactivity was detected in fetal and maternal blood vessel tissues during implantation and throughout gestation, and in preimplantatory trophoblast cells and uterine epithelium. After implantation the immunoreaction was confined to TGC and uterine epithelium. An antibody against bovine VEGF revealed a strong reactivity in the stroma of maternal caruncular septa in early and mid-gestation, which distinctly decreased near term. In interplacentomal areas, VEGF was found in luminal and glandular epithelia as well as in trophoblast, with distinctly higher reactivity in giant cells. VEGFR-1 was observed in trophoblast and uterine epithelium around implantation. Later, in definite placentomes, VEGFR-1 was localized in TGC near the chorionic plate and in maternal endothelial cells in the center of the placentome. VEGFR-1 and VEGFR-2 were co-localized in uterine epithelium and trophoblast as well as in blood vessel tissue and uterine glands. The presence of VEGF, VEGFR-1 and VEGFR-2 at the feto-maternal interface and in vasculature indicates that in the bovine VEGF may have (1) classic functions in angiogenesis and vascular permeability, (2) growth factor properties, facilitating feto-maternal exchange via paracrine action, (3) chemotactic activity on capillary endothelium, and (4) an autocrine influence on TGC migratory activity.

Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system.

Oxygen consumption is a useful parameter for evaluating embryo quality, since it provides a valuable indication of overall metabolic activity. Over the years, several approaches have been used to measure the respiration rates of individual embryos, but a convincing method has not yet been reported. In this study, we introduce and have validated a novel high resolution microsensor technology to determine the respiration rates of individual embryos at different developmental stages. We have employed this technology to investigate the correlation between respiration rate and embryo morphology, diameter and sex. Following morphological evaluation, individual respiration rates of day 3 (n = 18) and day 7 (n = 60) bovine in vitro-produced embryos were determined. Of the measured embryos, 64 were lysed for sex diagnosis by PCR. Average respiration rates of day 7 embryos (1.30 +/- 0.064 nl/h) were 3.4-fold higher than day 3 embryos (0.38 +/- 0.011 nl/h). On day 7, the average respiration rate of quality 1 blastocysts was significantly higher than the respiration rates of the lower qualities. For both day 3 and day 7 embryos, respiration rates were directly influenced by embryo diameter but did not differ between sexes. These results have demonstrated that the novel microsensor technology can be used to accurately and rapidly (8 min) measure the respiration rates of individual embryos at different developmental stages. Respiration rates were only in partial agreement with embryo morphology, suggesting a slight discrepancy between these two methods in assessing embryo quality. It is likely that a combined assessment of embryo respiration and morphology would improve embryo classification and subsequent selection.

Embryo technology: implications for fertility in cattle.

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

Hand-made cloning approach: potentials and limitations.

Two major drawbacks hamper the advancement of somatic cell nuclear transfer in domestic animals. The first is a biological problem that has been studied extensively by many scientists and from many viewpoints, including the cell, molecular and developmental biology, morphology, biochemistry and tissue culture. The second is a technical problem that may be responsible for 50% or more of quantitative and/or qualitative failures of routine cloning experiments and is partially the result of the demanding and complicated procedure. However, even the relatively rare documented efforts focusing on technique are usually restricted to details and accept the principles of the micromanipulator-based approach, with its inherent limitations. Over the past decade, a small alternative group of procedures, called hand-made cloning (HMC), has emerged that has the common feature of removal of the zona pellucida prior to enucleation and fusion, resulting in a limited (or no) requirement for micromanipulators. The benefits of HMC are low equipment costs, a simple and rapid procedure and an in vitro efficiency comparable with or higher than that of traditional nuclear transfer. Embryos created by the zona-free techniques can be cryopreserved and, although data are still sparse, are capable of establishing pregnancies and resulting in the birth of calves. Hand-made cloning may also open the way to partial or full automation of somatic cell nuclear transfer. Consequently, the zona- and micromanipulator-free approach may become a useful alternative to traditional cloning, either in special situations or generally for the standardisation and widespread application of somatic cell nuclear transfer.