The effect of dietary supplementation of algae rich in docosahexaenoic acid on boar fertility.

The objective of this study was to assess the effects of dietary supplementation of a commercial algal product rich in docosahexaenoic acid (DHA) on boar fertility as assessed in vitro and in vivo. Boars were fed one of three experimental diets for 19 weeks: (i) Control (Ctl) diet (n = 31), (ii) Ctl diet plus 75g All-G-Rich per day (n = 31) or (iii) Ctl diet plus 150g All-G-Rich per day (n = 30). Parameters assessed were (i) raw semen quality; volume, sperm concentration, total motility and morphology (ii) liquid semen quality; progressive motility, viability, hypotonic resistance and acrosomal integrity (iii) frozen-thawed semen quality; motility, thermal stress, viability, membrane fluidity and mitochondrial activity (iv) sperm and seminal plasma (SP) fatty acid composition (FAC) (v) total antioxidant capacity (TAC) of SP and (vi) farrowing rates and litter sizes of sows (n = 1158) inseminated with liquid semen. Boars consuming 75g All-G-Rich had a larger semen volume (P < 0.05) and a higher total sperm number (P < 0.01) than the Ctl treatment, however, there was no effect of treatment on any other semen quality parameter (P > 0.05). There was no effect of dietary treatment on the FAC and TAC of SP or on farrowing rate and litter size (P > 0.05). There was an effect of dietary treatment on the FAC of sperm, represented by an 1.72 and 1.60 fold increase in the DHA content for 75 and 150g treatments, respectively, compared to the Ctl treatment. In conclusion, a significant increase in semen volume and total sperm number in boars supplemented 75g All-G-Rich daily, resulted in an increase in production of 3 to 4 more doses per ejaculate, thus, indicating that the feeding regime described within this study has the potential for increasing the output of boar studs.

Successful treatment of neonatal hemochromatosis as gestational alloimmune liver disease with intravenous immunoglobulin.

Neonatal hemochromatosis (NH) is a rare, often fatal disorder characterized by liver failure and hepatic and extrahepatic iron overload. Clinical manifestations can occur in utero or immediately after birth. Evidence suggests that most cases are due to a gestational disease with transplacental transfer of maternal IgG antibodies targeting the fetal liver resulting in immune injury. The alloimmune target is believed to be a fetal hepatocyte cell surface antigen, with subsequent complement activation resulting in severe loss of hepatocytes and fetal iron overload. This cascade of events leads to acute liver failure and neonatal death. With gestational alloimmune liver disease (GALD) being the mechanism of liver injury in most cases of NH, a new paradigm of treatment with intravenous immunoglobulin (IVIG) and exchange transfusion has been successfully used. We describe an extremely ill newborn with NH successfully treated with three doses of IVIG.

Effects of diet type on establishment of pregnancy and embryo development in beef heifers.

The objectives were to determine the effects of elevated blood urea concentrations on: (i) the response to superovulation, fertilisation rate, and early embryonic development in beef heifers, and (ii) embryo survival from days 7 to 35 of gestation. In Experiment 1, heifers (18-24 months) were allocated at random (n=20 per treatment) to one of the following diets: (i) ad libitum grass silage plus 5 kg commercial beef concentrates per day (controls); (ii) ad libitum grass silage plus 5 kg concentrates and 250 g feed grade urea per day (HE/HU); or (iii) ad libitum wheaten straw plus 250 g feed grade urea and 50 g vitamin/mineral mix per day (LE/HU). Serum urea concentrations were monitored throughout the experiment. Oestrus in heifers was synchronised using an intravaginal releasing device (CIDR(®), InterAg, New Zealand). Oestrus was detected and in vitro produced blastocysts (day 7, morphological grades 1 and 2) were transferred to the heifers 7 days later (19 days after start of treatment diets). The heifers were maintained on the dietary treatments for a further 28 days, when pregnancy status was determined by transrectal ultrasonography. Detected pregnancies were terminated using 15 mg luprostiol and recycled for Experiment 2. In Experiment 2, following a 14-day dietary rest period, the heifers were re-allocated at random to the three dietary treatments above. Heifers were treated with a CIDR for 8 days and 15 mg luprostiol was given 12h before pessary withdrawal. They received 144 mg pFSH (Folltropin(®)-V, Vetrepharm, Canada) given as 8 injections over 4 days commencing on day 6 of CIDR/dietary treatment. Heifers were artificially inseminated 48 h after progesterone pessary withdrawal using commercial semen of proven fertility by a competent inseminator. The heifers were maintained on their diets until slaughter, 3 days post insemination when corpora lutea numbers were determined and embryos were recovered and cell numbers determined visually. Serum urea concentrations were greater in heifers on LE/HU than in those on HE/HU diets, which in turn were greater than controls (7.1 ± 0.5, 4.9 ± 0.3 and 3.2 ± 0.1 mmol/L, respectively; P<0.05). There was no effect of diet type on pregnancy rate at day 35 (42%, 47% and 46%) and on the number of corpora lutea following superovulation (5.2 ± 0.8, 5.8 ± 1.5 and 6.8 ± 1.1) for heifers on control, HE/HU and LE/HU diets, respectively. The total number of embryos recovered per heifer was not different between the three groups (2.7 ± 0.6, 3.4 ± 1.1 and 4.8 ± 0.8 for heifers on control, HE/HU and LE/HU diets, respectively; P>0.05), but the number of embryos with 8 or more cells at recovery was greater in heifers on LE/HU than on control diets (3.4 ± 0.8 compared with 1.0 ± 0.3; P<0.05). However the percentage of embryos recovered with 8 or more cells was not different between groups (70.0 ± 13.3, 86.9 ± 7.2 and 76.5 ± 7.9%, for heifers on control, HE/HU and LE/HU diets respectively). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the heifers on the control diet than in the other two dietary treatments (61.3 ± 11.8, 92.0 ± 3.5 and 86.8 ± 5.4% for heifers on control, HE/HU and LE/HU diets, respectively; P<0.05). Deleterious effects of urea on reproduction were not found, suggesting that adverse effects of urea are likely to take place at the early oocyte development stage prior to ovulation or fertilisation following an increase in protein intake.

Progesterone-regulated changes in endometrial gene expression contribute to advanced conceptus development in cattle.

The postovulatory rise in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our study objective was to determine how elevated P4 alters endometrial gene expression to advance conceptus development. Synchronized heifers were inseminated (Day 0) and randomly assigned to pregnant high P4 or to pregnant normal P4. All high P4 groups received a P4-release intravaginal device on Day 3 after insemination that increased P4 concentrations up to Day 7 (P < 0.05). Tissue was collected on Day 5, 7, 13, or 16 of pregnancy, and endometrial gene expression was analyzed using the bovine Affymetrix (Santa Clara, CA) microarrays. Microarray analyses demonstrated that the largest number of P4-regulated genes coincided with the day when the P4 profiles were different for the longest period. Genes with the largest fold change increase (such as DGAT2 and MSTN [also known as GDF8]) were associated with triglyceride synthesis and glucose transport, which can be utilized as an energy source for the developing embryo. Temporal changes occurred at different stages of early pregnancy, with the greatest difference occurring between well-separated stages of conceptus development. Validation of a number of genes by quantitative real-time PCR indicated that P4 supplementation advances endometrial gene expression by altering the time (FABP, DGAT2, and MSTN) or duration (CRYGS) of expression pattern for genes that contribute to the composition of histotroph.

The effect of feeding propylene glycol to dairy cows during the early postpartum period on follicular dynamics and on metabolic parameters related to fertility.

Postpartum dairy cows (n=35) were used to determine the effects of feeding propylene glycol (PG) on metabolic variables related to ovarian function and on oocyte developmental competence. Starting on Day 7 postpartum, each animal received an oral dose (500 ml) of either PG or water once daily. Blood samples were collected on Days 5, 15, 25 and 35 pp to measure insulin, non-esterified fatty acids (NEFAs), beta-hydroxybutyrate (BHB) and glucose concentrations. Oocytes were recovered by ultrasound guided follicular aspiration starting on approximately Day 30 postpartum and submitted to in vitro fertilization. Ovarian follicular activity was examined daily by ultrasonography from Day 7 until ovulation or Days 35-40 postpartum. Animals receiving PG had elevated insulin concentrations over the subsequent 90 min following dosing (P<0.05) compared to control animals. Glucose concentrations followed a similar pattern. Irrespective of treatment, concentrations of NEFA declined significantly from Days 15 to 35 postpartum. Administration of PG resulted in a decrease in NEFA (P<0.001) and BHB (P<0.001) over the subsequent 90 min compared to control animals. Treatment with PG had no effect on follicular dynamics, mean days to emergence of the first cohort of follicles postpartum, or days to dominance and duration of dominance for any follicular wave recorded postpartum. There was also no difference in mean days to first ovulation or in size of the preovulatory follicle between treatments. Oocyte quality as measured by blastocyst development after IVF was not affected by treatment. These results suggest that administration of PG has the ability to positively alter the systemic concentrations of a number of metabolic variables which have been related to fertility. However, we did not observe an effect of PG treatment on follicular dynamics or the length of the postpartum interval. An effect on oocyte developmental competence remains to be proven.

Induction of signalling in non-erythroid cells by pharmacological levels of erythropoietin.

Erythropoiesis is maintained by the hormone erythropoietin (Epo) binding to its cognate receptor (EpoR) on erythroid progenitor cells. The Epo-EpoR interaction initiates a signal transduction process that regulates the survival, growth and differentiation of these cells. Originally perceived as highly lineage-restricted, Epo is now recognised to have pleiotropic effects extending beyond the maintenance of red cell mass. Functional interactions between Epo and EpoR have been demonstrated in numerous cells and tissues. EpoR expression on neoplastic cells leads to concern that recombinant human erythropoietin, used to treat anaemia in cancer patients, may augment tumour growth. Here we demonstrate that EPO, at pharmacological concentrations, can activate three major signalling cascades, viz. the Jak2/STAT5, Ras/ERK and PI3K/Akt pathways in non-small cell lung carcinoma (NSCLC) cell lines. EpoR synthesis is normally under the control of GATA-1, but NSCLC cells exhibit decreased GATA-1 levels compared to GATA-2, -3 and -6, suggesting that GATA-1 is not essential for EpoR production. The increased Epo-induced signalling was not associated with a growth advantage for the NSCLC cells.

Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos.

In vivo-derived bovine embryos are of higher quality than those derived in vitro. Many of the differences in quality can be related to culture environment-induced changes in mRNA abundance. The aim of this study was to identify a range of mRNA transcripts that are differentially expressed between bovine blastocysts derived from in vitro versus in vivo culture. Microarray (BOTL5) comparison between in vivo- and in vitro-cultured bovine blastocysts identified 384 genes and expressed sequence tags (ESTs) that were differentially expressed; 85% of these were down-regulated in in vitro cultured blastocysts, showing a much reduced overall level of mRNA expression in in vitro- compared with in vivo-cultured blastocysts. Relative expression of 16 out of 23 (70%) differentially expressed genes (according to P value) were verified in new pools of in vivo- and in vitro-cultured blastocysts, using quantitative real-time PCR. Most (10 out of 16) are involved in transcription and translation events, suggesting that the reason why in vitro-derived embryos are of inferior quality compared with in vivo-derived embryos is due to a deficiency of the machinery associated with transcription and translation.

Alterations in follicular IGFBP mRNA expression and follicular fluid IGFBP concentrations during the first follicle wave in beef heifers.

The objective was to determine the pattern of IGFBP-2, -3 and -4 gene expression and follicular fluid concentrations of IGFBP-2, -3, -4 and -5 during emergence, selection and dominance of the first follicle wave of the estrous cycle in cattle and during exogenous steroid treatment. Heifers (n = 35) were ovariectomized at 36 (n = 7), 66 (n = 8), 84 (n = 12) and 108 (n = 8) h after the onset of estrus. Heifers in the 84 h ovariectomy group were sub-divided to receive either no treatment (n = 6) or were treated with a progesterone-releasing intravaginal device (n = 6, PRID) and 0.75 mg estradiol benzoate i.m. at the approximate time of ovulation, 30 h post estrus until ovariectomy. Within heifers the four largest follicles recovered following ovariectomy were ranked on size (F1, F2, F3 and F4). At 36 h IGFBP gene expression and follicular fluid IGFBP concentrations were similar in all follicles (F1-F4). Mean diameter of the F1 follicle increased (P < 0.05) between 36 and 84 h with no difference between 84 and 108 h. The F1 follicle had the highest (P < 0.05) concentration of estradiol compared with the F2, F3 and F4 at 84 and 108 h. There was no granulosa cell IGFBP-2 mRNA in F1 follicles at 84 or 108 h. Intrafolliclar IGFBP-2 concentrations were lower (P < 0.05) in the F1 compared with F3 and F4 follicles at 108 h. There was no difference in theca cell IGFBP-4 mRNA expression at 108h, but amounts of follicular fluid IGFBP-4 were lower (P < 0.05) in F1 follicles compared with F3 and F4 follicles at 108 h. IGFBP-3 mRNA was localized in the theca layer of all follicles examined with no difference in expression or follicular fluid concentrations during emergence, selection and dominance of the first follicle wave. IGFBP-5 concentrations were higher (P < 0.05) in follicular fluid of F3 follicles at 108 h compared with the F3 at 36 h. In conclusion follicular dominance was associated with low or decreased follicular fluid concentrations of IGFBP-4 and -5, increased estradiol and differential regulation of IGFBP production.

Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen.

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P<0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 x 10(6)/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination.

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.

The effects of ram exposure during progestagen oestrus synchronisation and time of ram introduction post progestagen withdrawal on fertility in ewes.

Three experiments were undertaken to investigate the effect of a pre-mating ram exposure during progestagen synchronisation treatment on time of breeding, ovulation rate, embryo quality and fertility and any interaction with time of ram introduction for breeding post sponge withdrawal. Crossbred ewes in experiment 1a (n = 348), 1b (mule; n = 133) and 2 (n = 58) underwent a 12-14 days synchronisation protocol. Three days prior to sponge withdrawal ewes were divided into Control (ewes in continued isolation from rams) or +Ram (ram-exposed) groups. Rams were introduced to +Ram ewes and remained with ewes until sponge withdrawal. Ewes in experiments 1a and 2 received eCG at sponge withdrawal and were reintroduced to rams at either 36 or 48 h post sponge removal (PSR). In experiment 1b, ewes did not receive eCG and were reintroduced to rams at 24 h PSR. In experiments 1a and 1b time of breeding, date of lambing and litter size were recorded. In experiment 2, ewes were slaughtered 5 days post breeding, reproductive tracts flushed and corpora lutea, ova and embryos assessed. Fewer +Ram ewes were mated by 96 h PSR (P < 0.001) than Control ewes in experiment 1a but not when rams were introduced earlier in experiment 1b. In experiment 1a, ram introduction at 36 h PSR improved conception to first service compared to introduction at 48 h PSR (P < 0.01) in both +Ram and Control groups. In experiments 1a and 1b, +Ram ewes had reduced litter size caused by more single births (1a; P < 0.001, 1b; P < 0.01). In experiment 2, +Ram ewes had fewer corpora lutea than Control ewes (P < 0.001) but embryo quality was similar. However, more good embryos were produced when rams were introduced for breeding at 36 h compared to 48 h PSR (P < 0.001). We conclude that a pre-mating ram exposure during the synchronisation treatment reduced the number of ewes mated at and conceiving to the first service. This was partially overcome by introducing rams for breeding earlier (24 or 36 h compared to 48 h PSR) but the most dramatic decrease in fertility was due to a reduction in ovulation rate in the ram-exposed ewes.

In vitro fertilization as a predictor of fertility from cervical insemination of sheep.

The objective of this study was to determine if the quality of frozen-thawed ram semen could be effectively evaluated through in vitro fertilization (IVF) procedures prior to insemination as a means of improving pregnancy rate. In experiment 1, frozen semen from four Belclare rams was assessed using IVF and was used for cervical insemination of ewes (n = 181) in 13 pedigree Belclare flocks. There was a significant association between IVF score (proportion of oocytes cleaved at 48 h post insemination) and non-return rate (P < 0.001). For experiment 2, semen from nine Belclare rams was evaluated by IVF and semen from rams with the highest (n = 3) and lowest (n = 2) IVF scores was used for cervical insemination of ewes (n = 111) under experimental conditions. Differences in pregnancy rates between individual rams did not reach significance. Experiment 3 was designed to determine if differences detected between rams at field level could be accurately identified via IVF evaluation and involved frozen semen from eight Norwegian rams of known field fertility (non-return rates ranged from 45.7 to 73.8%). IVF score did not reflect the differences in field fertility. In the final experiment six of the eight Norwegian rams involved in experiment 3 were selected based on IVF score (three highest and three lowest) and their semen was used for cervical insemination (n = 90 ewes). While significant differences in pregnancy rate were found between individual rams (P < 0.02, range: 12.9-65.8%) they were not associated with IVF score. Ewe breed had a significant effect (P < 0.003) on pregnancy rate in both experiments 2 and 4. In conclusion, there was no evidence from this study that the evaluation of semen quality through IVF provided a useful predictor of pregnancy rate under field conditions. It may be that the IVF procedures as used routinely, which are essentially designed to maximize blastocyst yields rather than for detecting differences in fertilizing ability between batches of sperm, need to be modified.

Effect of ram exposure at the end of progestagen treatment on estrus synchronisation and fertility during the breeding season in ewes.

Two experiments were conducted to examine the effects of ram exposure during the breeding season, in combination with progestagen treatment on estrus synchronization, fertility the LH surge and ovulation in ewes. Experiment 1 was subdivided into experiments 1a and 1b. In all experiments cross-bred ewes were treated with an intravaginal sponge for 12-14 days and three days before sponge withdrawal ewes were divided into control (no further treatment; n=191, 103 and 50 for experiments 1a, 1b and 2, respectively) or ram exposed (three mature rams per 50 ewes were introduced; +Ram; n=187, 99 and 49 for experiments 1a, 1b and 2, respectively). At sponge withdrawal ewes in Experiments 1a and 2 received 500 IU eCG and rams were removed from all the +Ram groups. In Experiments 1a and 1b, raddled, entire rams were introduced to ewes 48 h after sponge withdrawal. The timing of mating was recorded and ewes were maintained until lambing. In Experiment 2, estrus behavior was determined every 4 h and the time of the LH surge and ovulation were determined from a subset of 10 ewes per group. In Experiment 1a, less +Ram ewes were bred by 48 h after ram introduction (control 98% versus +Ram 89%, P<0.001) and in Experiments 1a and 1b 14% fewer (P<0.05) of the ewes bred in the first 3 h after ram introduction lambed to that service. In Experiment 1a, ram exposed ewes had a lower litter size than control ewes (1.93+/-0.06 versus 1.70+/-0.06 lambs per ewe; P<0.05). In Experiment 2, rams advanced (P<0.05) estrus, the LH surge and ovulation by 2-6 h compared with control ewes. We speculate that exposure of ewes to rams increased LH secretion and that this in turn increased follicle development and the production of oestradiol that led to a more rapid onset of estrus, the LH surge and ovulation compared to control ewes. Unexpectedly, ewes that were bred had lower fertility in the +Ram groups than control groups.

Fertility in the ewe following cervical insemination with fresh or frozen-thawed semen at a natural or synchronised oestrus.

Artificial insemination (AI) in sheep is currently limited by the poor fertility obtained following non-surgical intracervical insemination of frozen-thawed semen. An exception to this general finding is the non-return rate of around 58% reported for large scale on-farm AI in Norway. The objective of the present study was to determine if similar results could be obtained under Irish conditions. Comparisons were made between semen collected, and frozen, from rams in Norway (NOR) and Ireland (IRL). The effects of synchronisation and inseminator were also examined. Parous ewes (n=297) of various breed types were inseminated to a natural (N) or synchronised (S) oestrus with either fresh (from Irish rams) or frozen-thawed (IRL and NOR) semen. Ewes were randomly assigned, within breed, to the following treatment groups: (i) Fresh-N: n=28, (ii) Fresh-S: n=30, (iii) IRL-N: n=62, (iv) IRL-S: n=50, (v) NOR-N: n=68, (vi) NOR-S: n=59. Within each group, ewes were inseminated by an experienced Norwegian or by an Irish inseminator. Pregnancy rate did not differ significantly between ewes inseminated to a natural or synchronised oestrus nor between Norwegian and Irish frozen semen. The proportion of ewes pregnant after insemination with fresh semen was 0.82 and 0.70 (treatments i and ii) compared with 0.40, 0.52, 0.34 and 0.37 (treatments (iii)-(vi)) for frozen semen (P<0.001). Corresponding litter sizes (+/-S.E.), adjusted for ovulation rate, were 2.9+/-0.22, 3.3+/-0.23, 2.2+/-0.21, 1.7+/-0.21, 2.2+/-0.21 and 2.1+/-0.21 (fresh versus frozen; P<0.001). There was an interaction between semen type (fresh or frozen) and oestrus type (N or S) for litter size due to an increased adverse effect of frozen semen on litter size in synchronised ewes (P<0.05). Pregnancy rate was significantly influenced by breed of ewe (P<0.01) and inseminator (P<0.05). These results suggest that ewe breed may be a critical determinant of the potential for the exploitation of cervical insemination of frozen-thawed semen in sheep breeding programmes.

Effect of speed of development on mRNA expression pattern in early bovine embryos cultured in vivo or in vitro.

Recent data have demonstrated that fast-cleaving embryos produced in vitro are more likely to develop to blastocyst stage, and that the postfertilization culture system used impacts considerably on the mRNA expression and quality of blastocysts produced. The present study is the first to investigate the relationship between the developmental speed of embryos produced in vivo or in vitro and the temporal transcription pattern. Genes related to important preimplantation events are monitored during the first 4 days of embryo development in embryos with fast or slow development. The set of genes analyzed in the present study characterizes several important physiological processes including: transport and metabolism of fructose (Glut-5), stress (SOX), mitochondrial activity and detoxification of reactive oxygen species (MnSOD), cell communication (Cx43), maternal recognition of pregnancy (IFN-tau), imprinting (IGF-II), apoptosis (Bax), growth factor binding and metabolism (IGF-IR), and oxidative stress (G6PD). Using real time PCR, we have found that for all the genes analyzed there are differences in mRNA expression between embryos with fast and slow developmental speed produced both in vitro and in vivo. Frequently, genes that may be stress induced such as SOX, MnSOD, BAX, IFtau, and G6PD were highly transcribed in in vitro produced embryos and in embryos with slow developmental speed. On the other side, transcripts from genes related with metabolism, growth, and differentiation (Glut-5, Cx 43, IGF-II, and IGF-IR) were detected in higher amounts in in vivo produced embryos and in embryos with fast developmental speed. Moreover, it is interesting to stand out that for some genetic markers (such as SOX and G6PD) there are in vivo and in vitro differences that can be observed even before materno-zygotic transition, which probably reflects a differential mRNA degradation. These transcription patterns reflects the embryonic response to the adverse in vitro culture conditions, and connect the low quality of embryos which slow developmental speed produced in vivo and in vitro, with the mRNA expression pattern of some embryonic genes.

Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality.

The aim of this study was to examine the temporal sensitivity of bovine embryos to culture environment after fertilization to determine which period, if any, is most critical in determining blastocyst quality. Bovine zygotes produced in vitro were divided into six groups and cultured either in vitro (in synthetic oviductal fluid, SOF), in vivo (in the ewe oviduct) or in a combination of both systems. Development to the blastocyst stage, the ability of the blastocysts to withstand cryopreservation and the relative abundance of several gene transcripts were examined. Culture in SOF for either 2 or 4 days, followed by subsequent culture in the ewe oviduct, resulted in a significantly lower yield of blastocysts than did all other methods, the effect being most marked in embryos that were cultured in SOF for 4 days. In contrast, culture in vivo for the first 2 or 4 days after fertilization followed by culture in vitro did not have such a marked effect on blastocyst development. Blastocysts produced after culture in the oviduct for 6 days had the highest rates of survival over 72 h after warming (100% survival at 24 h; >95% survival at 72 h). The embryos that spent the last 4 days of culture in vivo also had relatively high rates of survival (100% at 24 h, 73.7% at 72 h). Blastocysts produced entirely in SOF had very low rates of survival after vitrification, with <40% viable at 24 h and <20% survival at 72 h. Blastocysts derived from embryos that spent the first 2 days in vivo and the last 4 days in vitro had the lowest rates of survival (6.7%), whereas those that spent the last 2 days only in SOF had intermediate rates of survival (40.6%). These differences were reflected in the relative abundance of transcripts for the Bax gene.

Oocyte and embryo quality: effect of origin, culture conditions and gene expression patterns.

In general, the majority of immature bovine oocytes fail to develop to the blastocyst stage following maturation, fertilization and culture in vitro. The evidence suggests that while culture conditions during in vitro embryo production can impact on the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. In addition, evidence suggests that the period of post-fertilization embryo culture is the most critical in determining blastocyst quality. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of oocyte origin and/or in vitro maturation conditions on the developmental capacity and gene expression patterns in the oocyte. Furthermore, the well-documented effects of post-fertilization culture environment on embryo gene expression and quality are highlighted.

Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo.

The objective of this study was to examine the time during the postfertilization period that gene expression patterns in in vitro-cultured bovine embryos diverge from those of their in vivo-cultured counterparts. Presumptive bovine zygotes were produced by in vitro maturation and fertilization of immature oocytes collected from the ovaries of slaughtered animals. Approximately 20 h post insemination (hpi), zygotes were denuded and randomly divided into two groups for culture either in vitro, in synthetic oviduct fluid medium, or in vivo, in the ewe oviduct. Embryos were recovered from both systems at approximately 30 hpi (2-cell), 2 (4-cell), 3 (8-cell), 4 (16-cell), 5 (early morula), 6 (compact morula), or 7 (blastocyst) days post insemination. On recovery, they were examined for stage of development and snap frozen in liquid nitrogen for the analysis of transcript abundance using real-time polymerase chain reaction. The transcripts studied were glucose transporter 5, sarcosine oxidase, mitochondrial Mn-superoxide dismutase, connexin 43, interferon tau, insulin-like growth factor II, apoptosis regulator box-alpha and insulin-like growth factor-I receptor, most of which are known from our previous work to differ in terms of transcript abundance in blastocysts derived from culture in vitro or in vivo. The results demonstrate that the relative abundance of the transcripts studied varies throughout the preimplantation period and is strongly influenced by the culture environment. In addition, the data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident by as little as 10 h of initiation of culture. Such information has implications not only for basic biology but also for human assisted reproduction in which there is a move toward culturing embryos to the blastocyst stage, necessitating prolonged culture in vitro under potentially deleterious conditions.

Influence of nutrition on the effectiveness of superovulation programmes in ewes: effect on oocyte quality and post-fertilization development.

Two experiments were carried out to study the effect of nutrition on embryo development in two periods in superovulated ewes (Expt 1) and on oocyte developmental capacity during the late follicular phase (Expt 2). In Expt 1, a lower superovulation response in terms of animals ovulating (P < 0.05), ovulation rate per ewe ovulating (P = 0.1) and number of good quality embryos per animal treated (P < 0.07) was noted in ewes fed an ad libitum diet compared with ewes offered control (1.5 times the daily maintenance energy requirements, 1.5 x M) or low energy (0.5 x M) diets. Nutrition also modified the morphological and functional quality of the oocytes and embryos recovered. Thus, 92% of day 4 embryos recovered from ewes offered the control diet were classified as good embryos, compared with 70 and 82% of those recovered from ewes offered the ad libitum and low diets, respectively (P < 0.05). Ewes offered the ad libitum diet had a greater percentage of poorly developed embryos compared with ewes offered the control or low diets (P < 0.05). Ewes fed the low diet tended to have more non-fertilized oocytes than ewes offered the control diet (P = 0.09). Diet of recipient ewes to which good quality embryos were transferred on day 4 did not affect embryo quality, when assessed 12 days later (day 16 of pregnancy). However, recipient diet affected prostaglandin F(2alpha) (PGF(2alpha)) production in vitro, and uterine tissue that originated from recipient ewes on the low diet secreted more PGF(2alpha) relative to uterine tissue that originated from recipients on the control diet (P < 0.05). In Expt 2, fewer total (P < 0.05) and good quality (P < 0.01) oocytes and a lower percentage of good quality oocytes (P < 0.01) were obtained from superovulated ewes offered the ad libitum diet compared with ewes offered the low diet. In addition, cleavage rate tended to be higher (51 versus 35%, P = 0.09) in ewes offered the low diet compared with ewes offered the ad libitum diet. In conclusion, changes in diet can affect the quality of the oocyte and embryo in superovulated sheep. A lower superovulation response and a decrease in the quality of oocytes and embryos indicate that ad libitum diets are highly detrimental for superovulatory programmes when compared with low and control diets. In addition, the results from the present study indicate that a low energy diet during early embryo development increased the uterine production in vitro of PGF(2alpha) which could lead to a poor uterine environment thereby compromising the development of the embryo.

Effect of reducing sperm concentration during IVF on the ability to distinguish between bulls of high and low field fertility: work in progress.

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5 x 10(6)sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125 x 10(6)sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5 x 10(6)sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5 x 10(6)sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.