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Juvenile barramundi were fed one of six diets containing differing docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) levels. Fish were restricted fed on a pair-fed feeding regime to eliminate variability in feed intake, with two diets fed to satiety to examine the effects of fixed or variable feed rations on EFA requirements. Weight gain, feed intake, feed utilisation, and physical clinical signs were monitored. No effect of dietary DHA and EPA concentration, DHA:EPA ratio or total LC-PUFA level was observed on weight gain, growth rate, feed conversion ratio (FCR), survival or physical clinical health signs (P > 0.05). Satiety fed fish had higher feed intake, final weight, weight gain and growth rate compared to their respective restrictively fed treatments (P − 1 DHA without compromising growth or health status.

Artificial insemination (AI) is an important technique in all domestic species to ensure rapid genetic progress. The use of AI has been reported in camelids although insemination trials are rare. This could be because of the difficulties involved in collecting as well as handling the semen due to the gelatinous nature of the seminal plasma. In addition, as all camelids are induced ovulators, the females need to be induced to ovulate before being inseminated. This paper discusses the different methods for collection of camel semen and describes how the semen concentration and morphology are analyzed. It also examines the use of different buffers for liquid storage of fresh and chilled semen, the ideal number of live sperm to inseminate and whether pregnancy rates are improved if the animal is inseminated at the tip of the uterine horn verses in the uterine body. Various methods to induce ovulation in the female camels are also described as well as the timing of insemination in relation to ovulation. Results show that collection of semen is best achieved using an artificial vagina, and the highest pregnancy rates are obtained if a minimum of 150×10(6) live spermatozoa (diluted in Green Buffer, lactose (11%), or I.N.R.A. 96) are inseminated into the body of the uterus 24h after the GnRH injection, given to the female camel to induce ovulation. Deep freezing of camel semen is proving to be a great challenge but the use of various freezing protocols, different diluents and different packaging methods (straws verses pellets) will be discussed. Preliminary results indicate that Green and Clear Buffer for Camel Semen is the best diluent to use for freezing dromedary semen and that freezing in pellets rather than straws result in higher post-thaw motility. Preservation of semen by deep-freezing is very important in camelids as it prevents the need to transport animals between farms and it extends the reproductive life span of the male, therefore further work needs to be carried out to improve the fertility of frozen/thawed camel spermatozoa.

Contents Artificial insemination (AI) is one of the most widely used reproductive technologies, and there is considerably interest in commercializing this technology in camels. Storage of semen extender frozen (at −20°C) is of considerable interest to scientists working with camels, as transportation of diluents at refrigeration temperature is not always possible given the hot, arid and remote conditions that dromedary camels exist in. Therefore, this study was conducted to compare the fertility of fresh camel semen, after dilution in fresh or frozen-thawed green buffer (GB), after AI into single and multiple ovulating female camels. No differences were observed in any sperm characteristics (motility, membrane integrity, acrosome integrity or morphology) when semen was diluted in fresh or frozen-thawed GB (p > 0.05). Sperm motility was increased by dilution (fresh: 70.7 ± 4.9% and frozen: 68.8 ± 3.1%) compared with the motility of sperm in neat semen (35 ± 2.85%; p

Sperm sexing is an emerging reproductive technology which has been successfully used to produce offspring of a pre-determined sex in domestic and wildlife species but has yet to be applied to New World camelids. The aims of the present study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y chromosome-bearing alpaca (Vicugna pacos) sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X- and Y-chromosome and (iii) determine the DNA difference between X- and Y-bearing sperm in alpacas. Semen was collected from alpacas and sperm nuclei stained with H33342, incubated and analysed using a high-speed cell sorter (SX-MoFlo). H33342 staining concentrations of 36, 54, 72 or 90 microm did not affect the proportion of correctly oriented sperm nuclei (43.3 +/- 3.9, 46.4 +/- 3.7, 44.5 +/- 4.0 and 51.1 +/- 2.5% respectively) nor the speed of sorting (1381 +/- 160, 1386 +/- 123, 1371 +/- 133 and 1379 +/- 127 sperm nuclei/s). Sort reanalysis determined high levels of purity for X- and Y-enriched populations (96.6 +/- 0.7% and 96.1 +/- 1.1% respectively). The DNA difference, based on fluorescence intensity (determined by the SX-MoFlo), was 3.8 +/- 0.06%. These data demonstrate for the first time that alpaca sperm nuclei can be separated into high purity populations and the potential for applying sperm sexing technology to New World camelids.

The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embryos derived from adult ewes and from unstimulated (16- to 24-week-old) and hormone-stimulated prepubertal (3- to 5-week-old) ewes were assessed. Cleavage was lower for hormone-stimulated (617/1025; 60.2%) than unstimulated prepubertal (117/169; 69.2%) and adult ewe oocytes (184/267; 68.9%; P0.05). Blastocyst formation by Day 7 (from zygotes) was similar for unstimulated (45/117; 38.5%), hormone-stimulated prepubertal (229/617; 37.1%) and adult ewes (101/184; 54.9%). Blastocysts derived from hormone-stimulated prepubertal ewes developed mainly on day 7, compared with Day 6 for adult and unstimulated prepubertal ewes. Pregnancy rates (day 60) and embryonic loss (between Days 20 and 60) did not differ after transfer to adult recipient ewes of adult, unstimulated and hormone-stimulated prepubertal-derived fresh or frozen-thawed embryos. The number of lambs born as a proportion of embryos transferred did not differ for fresh and frozen embryos derived from adult ewes (3/16; 18.8% and 1/12; 8.3%, respectively) and unstimulated prepubertal lambs (2/6; 33.3%, and 1/10; 10.0%, respectively), but was higher for fresh than frozen embryos from hormone-stimulated prepubertal ewes (7/16; 43.8%, and 2/14; 14.3%, respectively; P0.05). There were high rates of in vitro and in vivo development of oocytes from 3- to 5-week-old lambs, but in vitro development was lower than with oocytes from adult ewes. However, the speed of embryonic development in vitro and the in vivo development of fresh and frozen embryos were similar to those derived from adult and unstimulated prepubertal ewes. The present results were an improvement in the efficiency of producing embryos and offspring from hormone-stimulated 3- to 5-week-old lambs.

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.

Sex-sorting of mammalian spermatozoa has applications for genetic improvement of farm animals, in humans for the control of sex-linked disease, and in wildlife as a captive management strategy and for the re-population of endangered species. Considerable research has been undertaken worldwide on the Beltsville sperm sexing technology, the only effective method for pre-selection of sex of offspring. The combination of this method with assisted reproductive technologies has resulted in the birth of offspring in a wide range of animals, including cattle, the only livestock species in which sperm sexing is used commercially. Major improvements in the efficiency of sorting, in particular the development of high speed sorting (15 million X and Y spermatozoa per hour) have led to the production of offspring using conventional and low dose AI and the successful cryopreservation of sorted spermatozoa in cattle, sheep, horses and elk. A major limitation remains the short viable lifespan of sorted spermatozoa in the female genital tract, in most species necessitating sperm deposition deep in the uterus, and close to the expected time of ovulation, for acceptable fertility after in vivo insemination. Special deep uterine insemination technology has been employed to produce offspring in pigs and horses using low sperm doses. Considerable attention has been paid to reduction of the damage and capacitation-like changes to spermatozoa that result from flow cytometric sorting and from freezing and thawing. However, high-purity sorting of liquid-stored or frozen-thawed spermatozoa for immediate use, or re-cryopreservation for later use, does not reduce its fertilizing capacity in vitro, allowing its combination with in vitro fertilization or juvenile in vitro embryo transfer to produce blastocysts, and offspring in sheep and cattle after embryo transfer. Further research into sorting and preservation methods that incorporate strategies to prevent destabilization of sperm membranes may improve the fertilizing lifespan of flow cytometrically sorted spermatozoa. With continued improvement in sorting instrumentation and biological handling, sorting efficiency should reach a point where commercially acceptable pregnancy rates may be achieved in a number of species after conventional or deep uterine insemination.

The reproductive efficiency of camels is low under natural pastural conditions and so the use of artifical insemination and embryo transfer are becoming increasingly important to improve their breeding potential. Methods to control their reproductive cycle are therefore essential. This review describes characteristics of the ovarian follicular wave pattern in camels and exogenous hormonal control of ovulation. It also summarizes the difficulties involved with artifical insemination and analyzing the highly gelatinous semen, and reports on the latest methods used to try and reduce the viscosity and liquefy camel semen. In addition an account is given of different hormonal and physical methods used to synchronise follicular waves, and various hormone treatments used to broaden the availability of ovulated, asynchronous and non-ovulated recipients are discussed.

The reproductive efficiency of camels is low under natural pastural conditions and so the use of artifical insemination and embryo transfer are becoming increasingly important to improve their breeding potential. Methods to control their reproductive cycle are therefore essential. This review describes characteristics of the ovarian follicular wave pattern in camels and exogenous hormonal control of ovulation. It also summarizes the difficulties involved with artifical insemination and analyzing the highly gelatinous semen, and reports on the latest methods used to try and reduce the viscosity and liquefy camel semen. In addition an account is given of different hormonal and physical methods used to synchronise follicular waves, and various hormone treatments used to broaden the availability of ovulated, asynchronous and non-ovulated recipients are discussed.

1. We have used selective tachykinin receptor agonists and antagonists to investigate the nature of the receptors mediating responses to endogenous and exogenous tachykinins in the rabbit iris sphincter preparation in vitro. 2. The NK1-selective agonist, substance P methyl ester, induced contraction with a pD2 of 9.16 indicating the presence of NK1 receptors. In confirmation, the NK1-selective antagonist, GR82334, competitively antagonized responses to substance P methyl ester with high affinity (pKB 7.46). 3. NK3 receptors also mediate contraction since NK3-selective agonists exhibited high potency, e.g. the pD2 of [Me-Phe7]-neurokinin B was 9.67, and their responses were not inhibited by GR82334 (10 microM). 4. NK2 receptor activation does not seem to contribute to contraction since the NK2-selective agonist [beta-Ala8]-neurokinin A(4-10) had relatively low potency (pD2 6.43), and the NK2-selective antagonists MEN10207 (1 microM) and L-659,877 (10 microM) were inactive or had low affinity, respectively. 5. GR82334 (1 microM) significantly inhibited responses to electrical field-stimulation of non-adrenergic non-cholinergic sensory nerves (3, 10 and 30 Hz), and caused a rightward shift of the log concentration-response curve to bradykinin (lateral shift ca. 1000 fold). Higher concentrations of GR82334 (10 microM) significantly attenuated responses to capsaicin (1-60 microM) whilst completely abolishing responses to field-stimulation (3, 10 and 30 Hz) and bradykinin (1 nM- 3 microM). 6. In conclusion, NK1 and NK3 receptor activation results in contraction of the rabbit iris sphincter. The contractile response following sensory nerve stimulation by bradykinin, capsaicin and electrical field stimulation results from NK1 receptor activation.

1. Bradykinin (BK) receptors of the guinea-pig taenia caeci were compared with those of the guinea-pig trachea, a preparation proposed to possess novel BK3 receptors. 2. Bradykinin-evoked contractile responses were unaffected in both preparations by the selective BK1 receptor antagonist [des-Arg9,Leu8]-BK (1 microM-10 microM). The BK2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]-BK, both had low affinities (apparent pKB estimates less than 6) which did not differ significantly between the two preparations (P greater than 0.05). In contrast, the novel bradykinin receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140) potently antagonized responses to bradykinin with relatively high affinity (apparent pKB = 8.42 +/- 0.15 and 8.94 +/- 0.16 in the taenia caeci, and trachea, respectively). 3. We conclude that the bradykinin receptors in the guinea-pig taenia caeci have similar recognition properties to those present in the guinea-pig trachea, and in this respect the taenia caeci represents a useful preparation for the further study of proposed novel BK3 receptors.

Artificial insemination (AI) is poorly developed in camelids owing to the difficulty in collecting high quality semen and the highly viscous nature of the semen. Semen collected by artificial vagina (AV) is often of low quality and must be improved before any further development of AI technology can occur. The present study investigated the effects of adding a cervix-like stricture to the AV, presence of females, collecting semen into Androhep, skim-milk or Tris diluents, and catalase supplementation (0, 100, 200 or 600 units/ml) of Tris diluent on alpaca semen quality parameters. The addition of a cervix-like stricture increased mating length (p0.05), whilst the presence of females during semen collection did not improve semen quality parameters (p0.05). Collection of semen into Tris diluent improved sperm motility (58.0 +/- 11.9%) compared with the control (34.0 +/- 10.8%; p0.05), Androhep (33.5 +/- 10.7%) and skim-milk diluents (28.2 +/- 10.4%). Semen viscosity was reduced by collection into Androhep (4.6 +/- 1.7 mm) and skim-milk diluents (3.6 +/- 1.3 mm) compared with Tris diluent (5.7 +/- 2.1 mm) and no collection medium (9.3 +/- 3.5 mm; p0.05). Tris diluent supplemented with 100, 200 or 600 units/ml catalase increased semen viscosity (5.0 +/- 3.2 and 4.9 +/- 3.2 mm). Collection of alpaca semen by AV into Tris diluent increased semen quality facilitating further development of AI technology in alpacas.

We have used novel selective agonist ligands to examine neurokinin receptors mediating the contractile response to tachykinins in the rabbit iris sphincter preparation in vitro. The selective NK-1 receptor agonist δ-amino valeryl-[L-Pro 9 ,N-Me Leu 10 ]SP-(7–11) (GR73632) and the NK-3 receptor-selective agonist succ-[Asp 6 ,N-Me-Phe 8 ]SP-(6–11) (senktide) were both very active (concentration range 0.032 pM–10 nM and 0.1 pM–32 nM respectively), and were 933 and 16.6 times more potent than substance P, respectively, in contracting the iris. In contrast, the NK-2 selective agonist [Lys 3 ,Gly 8 -R-γ-lactam,Leu 9 ]NKA-(3–10) (GR64349) was active only at the highest concentrations tested (3.2 nM–32 μM), and had 0.054 the activity of substance P. The presence of several peptidase inhibitors was without effect on the concentration-response relationship to substance P, GR73632, GR64349 or senktide. Tachykinins differed in their offset kinetics. Responses to GR73632, GR64349 and senktide were rapid in offset (times to reach half maximal responses were 1.5, 1.1 and 5.1 min, respectively), whereas responses to substance P were very much more prolonged in duration (time to reach half maximal response was 35.3 min). These results suggest the presence of both NK-1 and NK-3 receptors mediating contraction of the rabbit iris sphincter preparation. In addition, differences in response offset kinetics seem not to be due to differences in peptide metabolism, and suggest a property of substance P not shared by the other tachykinins used in this study.

1. This study investigated the recognition characteristics of neurokinin receptors mediating potentiation of the contractile response to field stimulation in the guinea-pig vas deferens. 2. A predominant NK1 receptor population is strongly suggested by the relative activities of the common naturally-occurring tachykinin agonists, which fall within less than one order of magnitude. This conclusion is supported by the relative activities of the synthetic NK1 selective agonists substance P methyl ester, [Glp6,L-Pro9]-SP(6-11) and delta-aminovaleryl-[L-Pro9,N-MeLeu10]- SP(7-11) (GR73632) which were 0.78, 9.3 and 120 as active as substance P, respectively. Furthermore, the NK2 selective agonist [Lys3, Gly8,-R-gamma-lactam-Leu9]-NKA(3-10) (GR64349) was active only at the highest concentrations tested (greater than 10 microM), and the NK3 selective agonist, succ-[Asp6,N-MePhe8]-SP(6-11) (senktide) was essentially inactive (10 nM-32 microM). 3. [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP(1-11) antagonized responses to neurokinin A, neurokinin B, physalaemin, eledoisin, [Glp6,D-Pro9]-SP(6-11), GR73632 and GR64349 (apparent pKB s 5.6-6.2), but was less potent in antagonizing responses to substance P, substance P methyl ester and [Glp6,L-Pro9]-SP(6-11) (apparent pKB s less than or equal to 5.0-5.0). 4. In contrast, the recently developed NK1-selective receptor antagonist [D-Pro9[Spiro-gamma-lactam]Leu10,Trp11]-SP(1-11) (GR71251) did not produce agonist-dependent pKB estimates. Schild plot analysis indicated a competitive interaction with a single receptor population where the antagonist had an estimated overall pKB of 7.58 +/- 0.13 for the four agonists of differing subtype selectivity tested (GR73632, GR64349, substance P methyl ester and neurokinin B). This estimate is similar to that we obtained for NK1-mediated (substance P methyl ester) contraction in the guinea-pig ileum preparation (pKB= 7.86+ 0.05). 5. Tachykinin action appears not to depend on release of a number of intermediary mediators including acetylcholine, histamine or cyclo-oxygenase products, nor to involve interaction with neuronal mechanisms including alpha 2-adrenoceptor feedback, noradrenergic Uptake-I or opioid-release, since antagonism or inhibition of these mechanisms did not modify responses to tachykinins. 6. We conclude that tachykinin action in the field-stimulated guinea-pig vas deferens preparation is mediated through interaction with a predominant neurokinin NK, receptor population and this preparation can therefore be used to study NK, modulation of sympathetic neurotransmission.

Two experiments were conducted to determine the effects of glycerol concentration and Equex STM paste on the post-thaw motility and acrosome integrity of epididymal alpaca sperm. In Experiment 1, epididymal sperm were harvested from male alpacas, diluted, and cooled to 4 degrees C in a Lactose cooling extender, and pellet-frozen in a Lactose cryodiluent containing final glycerol concentrations of 2, 3, or 4%. In Experiment 2, epididymal sperm were diluted in Biladyl, cooled to 4 degrees C, stored at that temperature for 18-24 h, and further diluted with Biladyl without or with Equex STM paste (final concentration 1% v:v) before pellet freezing. In Experiment 1, sperm motility was not affected by glycerol concentration immediately (2%: 16.1 +/- 4.6%; 3%: 20.5 +/- 5.9% and 4%: 18.5 +/- 6.6%; P0.05) or 3h post thaw (5% for all groups; P0.05). Post-thaw acrosome integrity was similar for sperm frozen in 2% (83.6 +/- 1.6%), 3% (81.3 +/- 2.0%) and 4% glycerol (84.8 +/- 2.0%; P0.05) but was higher 3h post-thaw for sperm frozen in 3% (75.7 +/- 3.8%) and 4% (77.2 +/- 4.1%) than 2% glycerol (66.9 +/- 2.7%; P0.05). In Experiment 2, sperm motility was higher immediately after thawing for sperm frozen in the presence of Equex STM (Equex: 21.5 +/- 3.5%; control: 14.4 +/- 2.1%; P0.05) but was similar at 3h post-thaw (P0.05). Acrosome integrity was similar for sperm frozen with or without Equex STM paste immediately (control: 89.6 +/- 1.2%; Equex: 91.1 +/- 1.4%; P0.05) and 3 h post-thaw (control: 69.3 +/- 3.7%; Equex: 59.9 +/- 5.8%; P0.05). Sperm cryopreserved in medium containing 3-4% glycerol and 1% Equex STM retained the best motility and acrosome integrity, even after liquid storage for 18-24 h at 4 degrees C prior to cryopreservation.

Oocytes from abattoir-sourced ovine ovaries were aspirated from 2- to 4-mm follicles using 25, 50 or 100 mmHg pressure and an aspiration pump, or a needle (20-G) and syringe (2.5 ml) and subjected to in vitro maturation, fertilization and culture to determine the effect of aspiration pressure on the number and quality of oocytes recovered, and early embryonic development. Oocyte recovery rate was similar between groups (range: 57.1-73.1%; p > 0.05). The number and percentage of grade I and II oocytes recovered was reduced for 100 mm (24.5 +/- 3.6 and 31.1 +/- 6.1%) compared with 25 mm (51.4 +/- 7.0 and 60.2 +/- 7.8%) and 50 mm pressure (40.8 +/- 5.6 and 50.3 +/- 4.4%) and a syringe (40.3 +/- 12.0 and 45.2 +/- 2.1%; p < 0.05). Oocyte cleavage was similar for all groups at 24 (range: 30.9-49.6%) and 48 h post-insemination (49.7-65.5%), but blastocyst formation (% cleaved oocytes) was lower for oocytes aspirated with 25 mm (37.8%) than 50 (69.2%) or 100 mm (67.2%) pressure, and a syringe (72.0%; p < 0.05). Embryo production efficiency (% of oocytes cultured developing to the blastocyst stage) was higher for oocytes aspirated with 50 mm (45.4%) and 100 mm pressure (43.8%) and a syringe (45.0%) than 25 mm pressure (18.8%; p < 0.05). These results demonstrate that the aspiration of ovine oocytes with an aspiration pressure of 50 mm, or aspiration with a needle and syringe are equally efficacious for the in vitro production of embryos.

Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4°C in citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in 0.25- and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4°C and after freeze–thawing (0 but not 3 h post-thaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P < 0.05). Post-thaw acrosome integrity after cooling to 4°C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactose- or Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for 0.25- or 0.5-mL straws on dry ice or liquid nitrogen vapour (P < 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P > 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended.

Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm. Mol. Reprod. Dev. 74: 931–940, 2007. © 2007 Wiley-Liss, Inc.

Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (>23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.

Ovum pick-up (OPU) was performed three times on adult ewes after synchronization with (n = 4) or without (n = 4) FSH treatment to investigate the effects of FSH treatment on the number of ovarian follicles, oocytes recovered, oocyte quality and development in vitro. FSH treatment increased the number of ovarian follicles (85 vs 162) and oocytes recovered (33 vs 91), although recovery rate was similar for ewes with and without FSH (91/162, 56.2% and 33/85, 38.8% respectively). Of the oocytes recovered, those classified as grades I and II were similar between ewes with (78/91, 85.7%) and without FSH treatment (27/33, 81.8%). The number of ovarian follicles was similar after repeated OPU for ewes treated with FSH, but for ewes not treated with FSH the number of ovarian follicles decreased with repeated OPU. The number of oocytes recovered decreased for FSH-treated ewes only, while the oocyte recovery rate and proportion of oocytes classified as grades I and II were not affected by repeated OPU. Oocyte cleavage (46/78, 58.9% and 19/24, 79.2%) and blastocyst formation (35/46, 76.1% and 12/19, 63.2% respectively) were similar for ewes with and without FSH treatment. The number of ovarian follicles varied between ewes (p < 0.05) although the number of oocytes recovered and oocyte development in vitro were similar between ewes.

Ovine IVP embryos were derived from frozen-thawed unsorted and frozen-thawed sex-sorted spermatozoa that had been refrozen and thawed. The embryos were bisected and cultured in vitro, or transferred to recipient ewes to determine their survival in vitro and in vivo. Oocyte progression to the blastocyst stage was similar for unsorted (97/232, 41.8%) and sex-sorted spermatozoa (113/286, 39.5%; P > 0.05). Embryo survival in vitro post-bisection was similar for demi-embryos derived from unsorted and sex-sorted sperm, and embryos bisected at the blastocyst and expanded blastocyst stage (P > 0.05). A higher proportion of recipient ewes were pregnant at Day 63 after transfer of two intact embryos derived from unsorted (17/21, 80.9%) than two demi-embryos derived from unsorted (5/15, 33.3%) or sex-sorted spermatozoa (7/17, 41.2%). The number of fetuses per original embryo at Day 63 did not differ among groups (unsorted intact: 23/42, 54.8%; unsorted demi: 7/15, 46.7%; sex-sorted demi: 10/17, 58.8%) and twin pregnancies were observed in all groups. Embryo survival to term was high, and was not significantly different among intact (unsorted: 22/42, 52.4%) and demi-embryos (unsorted: 4/15, 26.7%; sex-sorted spermatozoa: 7/17, 41.2%; P > 0.05). Dizygotic twins (n = 6 sets) were born after the transfer of two intact embryos derived from unsorted spermatozoa, but only singleton lambs resulted from the transfer of demi-embryos. In conclusion, bisected IVP embryos successfully developed into morphologically normal lambs. However, embryo survival to term was neither increased nor decreased by embryo bisection.

Publisher Summary This chapter discusses the kinin receptor agonists and antagonists, as well as evidence for receptor subtypes. The chapter discusses the proinflammatory actions mediated by kinin receptors and, in particular, critically analyzes the evidence for important roles for kinins in inflammation. Kinins play a pivotal role in the pathophysiology of an inflammatory disease. It was only in the past decade or so that convincing evidence emerged establishing that most of the actions of kinins are mediated via cell surface receptors. As a consequence of the discovery of potent, subtype-selective receptor antagonists, and through the cloning and expression of bradykinin (BK) receptor genes, it is known that at least two major G protein-coupled receptor subtypes, B 1 and B 2 , mediate most of the kinins' actions. In considering the range of putative roles for BK in physiology and pathophysiology, one may refer to an exhaustive and provocative recent review by Stewart, which catalogs an extensive list ranging through topics like pain, inflammation, edema, shock, rhinitis, and asthma. Some indications are given as to types of antagonist being used in various sorts of clinical trials. Peptide antagonists are more likely to be efficacious where they can be applied locally; for instance, in rhinitis, the treatment of burn pain and in some types of neurogenic inflammation. More stable and nonpeptide antagonists, which can be administered systemically, are more likely to be used in acute treatment of otherwise fatal conditions, such as shock and edema.

1. In the smooth muscle of the guinea-pig taenia caeci, bradykinin produces a relaxation followed by a contraction. In the presence of hexamethonium and guanethidine, both these phases of the response were insensitive to tetrodotoxin (100 nM), omega-conotoxin GVIA (100 nM) and ibuprofen (1 microM), suggesting that they are due to a direct action on the smooth muscle. 2. The B1 receptor-selective agonist, [des-Arg9]-BK (1-100 microM), was inactive in the taenia caeci, and the B1 receptor-selective antagonist, [Leu8,des-Arg9]-BK (1-10 microM), did not inhibit either phase of the bradykinin-induced response. The B2 receptor-selective antagonist, D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (Hoe 140) (30-300 nM), inhibited both the bradykinin-induced relaxation and contraction with a similar affinity (apparent pKB estimates of 8.5 +/- 0.1 and 8.4 +/- 0.1 respectively). 3. In a depolarizing high-K(+)-solution, bradykinin produced concentration-related contractions, though of diminished magnitude; but no relaxation was observed in such media. In Krebs solution, the Ca(2+)-activated K(+)-channel blocker, apamin (10 nM), abolished relaxant responses. These observations suggest that contraction results both from membrane potential-dependent, and membrane potential-independent, mechanisms; whereas relaxant responses result entirely from membrane potential-dependent mechanisms. Contractile responses obtained in the high K(+)-solution were inhibited by D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK with an apparent pKB value of 8.4 +/- 0.1. 4. In a Ca(2+)-free, EGTA-containing medium, relatively high concentrations of bradykinin (100 nM) produced transient contractions, suggesting that a component of the contractile response results from release of Ca2+ from an intracellular store. This intracellular Ca2+ store could be refilled in the presence of extracellular Ca2+. The B, receptor antagonist, [Leu8,des-Argj-BK (10 micro M), did not inhibit this bradykinin-induced contraction, whereas the B2 receptor antagonist, D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK(100 nM) markedly attenuated it (P0.001; n = 6).5. Bradykinin (10 nM- 100 micro M) significantly elevated tissue levels of total [3H]-inositol phosphates in the presence of Li?, after incubation with myo-[3H]-inositol. The B, receptor-selective agonist, [des-Argl-BK(100IM) did not stimulate [3H]-inositol phosphate formation, and the B, receptor-selective antagonist,[Leu8,des-Argl-BK, did not inhibit the formation of [3H]-inositol phosphates in response to a submaximal concentration of bradykinin (1I0 1M; P0.05). Two B2 receptor antagonists, D-Arg-[Hyp3,DPhe7]-BK and D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK, inhibited bradykinin-induced accumulation of total[3H]-inositol phosphates with apparent pKB estimates of 5.4 +/0 0.3 and 8.4 +/- 0.1, respectively.6. These data suggest that in the guinea-pig taenia caeci, the five aspects of the action of bradykinin studied (the relaxant and the contractile elements of the biphasic mechanical response, the contractile response in a depolarizing high-K' solution medium and zero-Ca2+ media, and stimulation of phosphatidylinositol turnover), all result from activation of B2 receptors. A possible causal relationship is suggested between these B2 receptor-mediated membrane potential-dependent, and -independent events,and their roles in excitation contraction coupling.

1. A contraction of the rabbit isolated iris sphincter smooth muscle follows activation of either tachykinin NK1 or NK3 receptors. We have here characterized the pharmacological activity profiles of various tachykinin receptor agonists considered to have NK1-receptor-preferring activity in this preparation. 2. Two groups of NK1-receptor-preferring agonists could be distinguished in terms of a common pharmacological profile. The first group (Group 1) included [Glp6,L-Pro9]-SP(6-11) (septide), [Glp6]-SP (6-11), substance P methyl ester, delta-aminovaleryl-[L-Pro9, N-MeLeu10]-SP(7-11) (GR73632), and [Apa9-10]-SP. The second group (Group 2) included [Pro9]-SP, substance P, physalaemin and ranamargarin. 3. Under control conditions, the responses to Group 1 agonists were relatively fast in offset (time for reversal of maximal responses, 11.2-18.2 min), and were antagonized by NK1-receptor-selective antagonists (range of pKB estimates vs various agonists; GR82334, 7.1-8.2; (+/-)-CP-96,345, 8.9-9.5; RP67580, 7.0-7.4). Following incubation of the tissue with phenoxybenzamine (20 microM, 10 min), the affinity of GR82334, tested against the Group 1 agonists, substance P methyl ester and septide, was not significantly different (P < 0.05; n = 7-18) to that determined in untreated tissues (substance P methyl ester pKB 7.5 +/- 0.1 and 7.2 +/- 0.2, respectively; septide 7.7 +/- 0.2 and 7.9 +/- 0.2, respectively). Further, response offset times (5.0-8.5 min) were little reduced as compared to those observed in untreated tissues. 4. Under control conditions, the response to Group 2 agonists was markedly slow in offset (times for reversal of maximal responses, 51.4-70.4min), and was not attenuated significantly by the NK1-receptor-selective antagonists GR82334 (I MicroM), (+/-)-CP-96,345 (0.1 MicroM) or RP67580 (1 MicroM). In contrast,after phenoxybenzamine pretreatment, responses to Group 2 agonists reversed rapidly (times for reversal of maximal responses, 13.1-24.2 min), and were now antagonized by GR82334 (pKB estimates, 6.4-7.1).5. The responses to the NK3-receptor-selective agonist Succ-[Asp6,Me-Phe8]-SP(6-l1) (senktide) were relatively fast in offset (time for reversal of maximal response was 18.6 +/- 1.7 min) and were not inhibited by GR82334 (10 MicroM; n = 5). The contractile response resulting from co-application of the Group 1 agonist, septide together with senktide, did not exhibit prolonged response offset kinetics.6. Assuming simple competition at equilibrium, these data from the rabbit iris smooth muscle could be explained either by interaction of the various ligands with two separately-existing NK1 receptor-subtypes or -isoforms; or alternatively by a preferential interaction of the two agonist groups with different binding domains on a common NK1 receptor.

1. We have estimated potencies of tachykinin receptor agonist and antagonist analogues in order to determine the recognition characteristics of tachykinin receptors mediating phasic contractile responses of the rat isolated urinary bladder in vitro. 2. The NK1-selective synthetic agonists, substance P methyl ester and GR73632, the synthetic NK2-selective agonists [beta-Ala8]-NKA(4-10) and GR64349, and the mammalian tachykinins, neurokinin A and neurokinin B, were assayed relative to substance P and were found to be approximately equipotent. The NK3-selective agonist, senktide, was inactive (10 microM). 3. Potencies of all these agonists were not significantly different (P > 0.05) when experiments were carried out in the presence of the neutral endopeptidase inhibitor, phosphoramidon, and the kininase II inhibitor, enalaprilat (both 1 microM). 4. The NK1-selective antagonist, GR82334, inhibited responses to substance P methyl ester in a competitive manner in the rat urinary bladder and the rat ileum, and also in the guinea-pig ileum. Markedly different pKB estimates were obtained in the rat bladder (6.38) and rat ileum (6.56) compared to the guinea-pig ileum (7.42). GR82334 (3 microM) was inactive against responses of the rat bladder to [beta-Ala8]-NKA(4-10). 5. The NK1-selective antagonist (+/-)-CP-96,345 also inhibited responses of the rat bladder and guinea-pig ileum to substance P methyl ester; however, in the rat bladder at 1 microM, this antagonist reversibly inhibited responses both to the NK2-selective agonist [beta-Ala8]-NKA(4-10) and to the muscarinic agonist carbachol (P < or = 0.01), thus showing evidence of some non-selective depressant actions. 6. The NK2-selective antagonists, MEN10207 and L-659,874, competitively inhibited responses of the rat bladder to the NK2-selective agonist [P-Ala5]-NKA(4-10) giving pKB estimates of 5.75 and 6.68,respectively. Both antagonists (1O microM) were inactive against responses to the NKI-selective agonist substance P methyl ester.7. These results support the proposal of a mixed population of NKI and NK2 receptors mediating contraction of the rat isolated urinary bladder. The NK2 receptor is characterized by a relatively low affinity for the NK2-selective antagonist MEN10207 but a high affinity for L-659,874. The NKImediated responses are inhibited by (+/-)-CP-96,345: this compound however, has non-specific depressant effects in the rat bladder at high concentration (1 microM). In contrast, the NK,-receptor peptide antagonist GR82334, did not have non-specific depressant effects and competitively inhibited NK, responses in the rat bladder and rat ileum with an affinity significantly lower than at the NK,-receptors in the guinea-pigileum.

This study investigated the subtype and coupling mechanisms mediating the direct contractile response to tachykinins in the guinea-pig Taenia caeci preparation in vitro. Coupling of neurokinin receptors was compared throughout with coupling of muscarinic receptors. The smooth muscle neurokinin receptors seem to be predominantly of the NK-1 subtype. Thus, the relative activities of the common naturally-occurring tachykinins fell within one order of magnitude, and the selective NK-1 receptor agonist substance P methyl ester was high in activity (0.38 relative to substance P). Some contribution from NK-3 receptors is, however, possible in view of the appreciable activity of the selective NK-3 agonist succ-[Asp6, N-McPhe8]-SP(6–11) (senktide; activity 0.004 relative to substance P), and NK-2 or NK-3 receptors in view of the higher activity of the D-isomer of [Glp6, *Pro9]-SP(6–11) as compared to its NK-1 selective L-isomer (D/L-activity ratio 1.53). Contractile actions of tachykinins were compared with carbachol for reliance on membrane-potential dependent (electromechanical) and membrane-potential independent (pharmacomechanical) coupling mechanisms. Log concentration-response curves to carbachol and substance P in normal Krebs' medium were compared with curves obtained in a high-K+ solution where processes dependent on changes in membrane potential could play no part in excitation. In the high-K+ depolarizing solution, a concentration-related relationship was maintained, though with some diminution in the maximal additional tension generated: the maximum tension with carbachol was under both conditions greater than that with substance P. The relative effects of several tachykinins and carbachol in producing receptor-mediated changes in membrane permeability through presumed receptor-operated ion channel opening, was estimated in terms of the ability to increase 86Rb-efflux, as a marker for K+, in a high-K+ depolarizing solution. Carbachol (10 μM) consistently increased 86Rb-efflux. In contrast, no permeability increase could be detected with any tachykinin tested (substance P, eledoisin, substance P methyl ester, neurokinin A, neurokinin B, 1 or 10 μM). Tachykinins and carbachol were compared in terms of ability to increase phosphatidylinositol hydrolysis. Both substance P and carbachol showed a concentration-related increase in accumulation of total inositol phosphates; though the maximal response to carbachol was considerably greater than that to any tachykinin (substance P, eledoisin, substance P methyl ester, senktide, neurokinin A, neurokinin B), or combination of two tachykinins (substance P and eledoisin, senktide and substance P methyl ester). Experiments in a high-K+ solution established that there was still an increase in accumulation of total inositol phosphates, thereby showing that this response was not a secondary consequence of membrane depolarization. Functional contractility studies, involving depletion of inositol pools in the presence of lithium (Li+), suggested a casual link between phosphatidylinositol turnover and contraction. These results are discussed in relation to similarities in coupling mechanisms between classical spasmogens (such as carbachol) and peptide spasmogens (such as tachykinins).

The effects of three different opioid agonists on contractions and [3H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine3 (5-HT3) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu (μ)-opioid receptor agonist (d-Ala2,NMe-Phe4,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa (κ)-opioid receptor agonist U50488 (10 nM -1 μM) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC50 estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta (σ)-opioid receptor agonist [d-Pen2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 μM). 3H-overflow from LMMP preparations preincubated with [3H]-choline was measured as an indicator of [3H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 μM) inhibited the increases in release of [3H]-ACh evoked by 5-HT (10 μM) and by senktide (10 nM) in a concentration-dependant manner. IC50 estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [3H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 μM). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [3H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 μM). These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT3 and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by μ- and κ-, but not σ-, opioid receptors.

We have investigated the receptors and associated coupling mechanisms that mediate the smooth muscle relaxant response to bradykinin (BK) in the rat duodenum in vitro. Relaxation in response to BK seems due to a direct action on the longitudinal smooth muscle since effects were demonstrable in the presence of ibuprofen, mepyramine, atropine, guanethidine (all 1 μM), hexamethonium (10 μM) and TTX (0.3 μM). Receptors involved are of the B 2 subtype since agonists and antagonists active at B 1 receptors were essentially inactive, and the B 2 receptor antagonist Lys,Lys-[Hyp 3 ,Thi 5,8 ,D-Phe 7 ]BK was a potent competitive antagonist of BK-induced relaxation (pK B of 7.2 ± 0.1). The activity of both BK and the antagonist were unchanged by the presence of peptidase inhibitors including the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (mergetpa, 10 μM), which prevents conversion of BK analogues to des-Arg 9 -B 1 -active products. In high-K + solution, BK (0.1–10 μM) produced concentration-related increases in 86 Rb efflux. Both this permeability increase in high-K + solution, and the relaxant responses in Krebs solution, were inhibited by low concentrations (10–100 nM) of apamin, as well as the B 2 receptor antagonist Lys,Lys-[Hyp 3 ,Thi 5,8 ,D-Phe 7 ]BK (1 μM). These results are compatable with the proposal that BK-evoked relaxation of the rat duodenum is mediated via a subset of B 2 receptors for which the antagonist Lys,Lys-[Hyp 3 ,Thi 5,8 ,D-Phe 7 ]BK has a high affinity, and results from stabilisation of the smooth muscle membrane through the opening of apamin-sensitive 86 Rb-permeable calcium-activated K + channels.

1. The relative contributions of two classes of 5-hydroxytryptamine (5-HT) receptor (5-HT2 and 5-HT3) to the contractile action of 5-HT, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT) and alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) were studied in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. Contractility studies were combined with an analysis of the effects of the three agonists on [3H]-acetylcholine ([3H]-ACh) release from preparations preincubated with [3H]-choline. 2. In contracting the LMMP, 5-HT was approximately one order of magnitude more active than 2-methyl-5-HT and alpha-methyl-5-HT, with relative activities for 5-HT: 2-methyl-5-HT: alpha-methyl-5-HT of 1.00: 0.13: 0.10. 3. Ketanserin (1 microM) was without effect on the concentration-response curves for concentration to 5-HT. 2-methyl-5-HT or alpha-methyl-5-HT, whilst ondansetron (GR38032F: 1 microM) produced a parallel rightward displacement of the upper part of the concentration-response curves to 5-HT and alpha-methyl-5-HT and of the entire curve to 2-methyl-5-HT. 4. In increasing the spontaneous release of [3H]-ACh from the LMMP, 5-HT was again approximately one order of magnitude more active than 2-methyl-5-HT and alpha-methyl-5-HT with relative activities for 5-HT: 2-methyl-5-HT: alpha-methyl-5-HT of 1.00: 0.19: 0.11. 5. Ondansetron (1 microM) greatly attenuated the increase in spontaneous [3H]-ACh release evoked by all three agonists. pKB estimates of 7.62 + 0.12 and 7.64 + 0.09 were obtained for ondansetron antagonism of 5-HT and 2-methyl-5-HT-evoked increases respectively. 6. These data suggest that the contractile action of 5-HT, 2-methyl-5-HT and a-methyl-5-HT in the guinea-pig ileum can, under these conditions, be accounted for largely in terms of 5-HT3 receptor activation. Estimates for pKB obtained with ondansetron are in accordance with those previously obtained from contractility studies in this preparation and these findings are discussed in terms of the postulated existence of subtypes of 5-HT3 receptors.

The influence of degradation by peptidases on concentration-response relationships for peptide agonists of the tachykinin and bombesin-like families was investigated. The combined presence of three peptidase inhibitors, phosphoramidon (1 microM), captopril (1 microM) and bestatin (100 microM), had no significant effect on the onset rates or peak contractile responses to these peptides in the rat urinary bladder and guinea-pig taenia caeci preparations, or on their peak potentiation of the contractile response to field-stimulation in the guinea-pig vas deferens preparation. However, rates of offset of the response to tachykinins were markedly prolonged in tissues treated with peptidase inhibitors. In experiments designed to estimate clearance of applied peptide from the organ bath, there was an initial rate of loss with the guinea-pig vas deferens and taenia caeci which, measured over the first 5 min, had a half-time of 2-3 min which was then prolonged to 6-8 min in the presence of peptidase inhibitors. These results show that although peptide breakdown can be demonstrated in these systems, it seems not to be an important determinant of relative pharmacological activity measured in terms of peak response.

The production of embryos from prepubertal lambs is inefficient, partly resulting from the low developmental competence of prepubertal lamb oocytes, and partly because a high proportion of lambs fail to respond to hormone stimulation. The development of a hormone stimulation regimen that all lambs respond to would increase the efficiency of breeding from prepubertal animals. Using a hormone stimulation regimen consisting of oestradiol benzoate (50 µg), a norgestomet implant (1.5 mg), pregnant mare serum gonadotrophin (400 IU) and follicle stimulating hormone (130 mg) all lambs (n = 19) responded to hormone stimulation. Uterine and ovarian weight ranged from 2.8 to 7.2 g (11.8 ± 0.7 g) and from 1.7 to 54.1 (12.5 ± 2.9 g), respectively. The number of ovarian follicles and oocytes recovered ranged from 20.0 to 500.0 (118.2 ± 29.2) and from 13.0 to 455.0 (82.0 ± 24.2), respectively, and oocytes suitable for in vitro production were obtained from all 19 lambs. Uterine weight was related to both bodyweight and growth rate (P < 0.05), although ovarian weight and the number of ovarian follicles were not related to either bodyweight or growth rate. Oocyte cleavage varied between hormone-stimulated lambs (0.0–93.0%; P < 0.05), and 484/775 (62.2%) of the oocytes cultured cleaved. Oocytes from 17 of the 19 lambs (89.5%) developed to the blastocyst stage in vitro, and the proportion of zygotes forming a blastocyst (by Day 7) ranged from 0.0 to 66.7% for individual lambs. Overall, 33.9% of zygotes (n = 164) developed to the blastocyst stage, producing 8.6 ± 2.8 blastocysts per lamb.

Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment 1) and response to stimulation (Experiment 2) on the in vitro production of embryos from prepubertal lambs aged 3–4 and 6–7 weeks of age. For 3–4-week-old lambs, hormone stimulation increased the number of follicles (29.9 ± 15.3 v. 70.6 ± 8.2), oocytes per ovary (18.3 ± 6.3 v. 39.3 ± 5.8) and oocyte development to the blastocyst stage (0/192 (0.0%) v. 115/661 (17.4%); P < 0.05). Lamb age (3–4 v. 6–7 weeks old) increased oocyte development to the blastocyst stage (115/661 (17.4%) v. 120/562 (21.4%) respectively). In Experiment 2, hormone-stimulated lambs (3–4 and 6–7 weeks old) were divided into low, medium or high responders based on the number of ovarian follicles (100 follicles per ovary respectively). The response to hormone stimulation did not affect oocyte recovery rate, but the number of oocytes suitable for culture was increased for high-responding 3–4-week-old lambs only (P < 0.05). Oocyte development to the blastocyst stage was not affected by response to stimulation for 3–4-week-old lambs (15.2–25.6%; P > 0.05), but was reduced for high (6.7%) compared with low (19.5%) and medium (30.9%) responding 6–7-week-old lambs (P < 0.05). These results demonstrate that the production of embryos from prepubertal lambs is increased by hormone stimulation and lamb age and the response to stimulation does not affect embryo production from 3–4-week-old lambs, although by 6–7 weeks of age a high response to stimulation reduces blastocyst formation.

Pre-sexed embryos and offspring have been produced after IVF and embryo transfer (ET) with sex-sorted frozen-thawed sperm in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95). The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a predetermined sex and that these embryos could be successfully nonsurgically transferred. Ovaries were collected from abattoir slaughtered gilts (n = 138) and selected COCs were matured in vitro (Long et al. 1999 Theriogenology 51, 1375–1390). Sperm were collected from a mature boar and diluted with Androhep (1:3, semen:Androhep; Minitube, Verona, WI, USA), stained with H33342, and separated into X and Y sperm using a SX MoFlo (Cytomation, Inc., Fort Collins, CO, USA). Sex-sorted sperm were cryopreserved in 0.5 mL straws using the Westendorf protocol modified for sorted sperm (Bathgate, unpublished). Thawed sperm (Y sperm only) were prepared for IVF by centrifugation (300g, 10 min) through a Porcipure gradient (Nidacon Int. AB, Gothenburg, Sweden), and washed (centrifugation 300g, 10 min) in mTALP-PVA. For IVF, COCs were denuded and groups of 100 oocytes were transferred to 200-μL drops of mTALP-PVA (Long et al. 1999) and incubated with 5,000 motile sperm for 4–6 (Short) or 18–20 h (Long) . Presumptive zygotes were washed and transferred to 100-μL drops of mNCSU-23 (Long et al. 1999) and cultured until Day 4 (Day 0 = IVF) in humidified 5% CO2, 6% O2, 89% N2. Oocyte cleavage was assessed 48 h post-insemination, and on Day 4 selected morulae were transferred to recipient sows (n = 7 Large White × Landrace; 65 morulae per sow) nonsurgically using a Firflex catheter (Magapor, Zaragoza, Spain). Sex of remaining embryos was confirmed by PCR and restriction analysis (Cong et al. 1993 Hum. Mol. Genet. 2 1187–1191). Data from three replicates were arc sin transformed and analyzed by ANOVA. Oocyte cleavage was similar after Short (724/1547; 46.8%) or Long (598/1528; 39.1%) co-incubation. Resort analysis showed sperm to be >91% purity, and all sexed morulae were of the predicted sex (16/16). Delayed return to estrus (>23 days) was observed in 5 recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine IVP and ET protocols are required to enable full in vivo development. This work was supported by XY, Inc., Fort Collins, CO, USA.

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

Developments in sperm sexing technology have resulted in the birth of a number of offspring after IVF of oocytes from adult animals (Johnson LA, 2000 An. Reprod. Sci. 60–61, 93–107). The aim of this study was to combine sperm sexing technology with juvenile breeding. Merino lambs, 2–3 weeks (n = 43) were hormone stimulated (Morton KM et al., 2003 Proc. Soc. Reprod. Fert., P18), and COCs were matured in TCM-199 (Sigma) with 10 μg mL−1 p-FSH (Folltropin-V; Bioniche Animal Health Australasia), 10 μg mL−1 pLH (Bioniche), and 20% sheep serum (v/v) in a humidified 6% CO2, 5% O2, 89% N2 atmosphere for 22 h. Semen collected from Merino rams was diluted and frozen as pellets (Unsorted), or stained with H33342, separated into X and Y sperm using a SX MoFlo (Cytomation Inc., Fort Collins, CO, USA), and frozen as pellets (Sorted). Sperm were prepared for IVF by swim-up under 0.5 mL of SOF with 2% sheep serum (v/v; SOF+) for 45 min (Unsorted), or diluted in 0.5 mL of Sydney IVF Sperm Buffer (Cook IVF, Brisbane, Australia) and centrifuged at 650g for 3 min (Sorted). After IVM, oocytes were transferred to SOF+, and cultured with 0.5 × 106 mL−1 (Unsorted) or 1.0 × 106 mL−1 (Sorted) motile sperm for 18 h. Presumptive zygotes were transferred to Sydney IVF cleavage and blastocyst medium (Cook IVF) for 3 and 5 days, respectively. Oocyte maturation and fertilization were assessed by orcein staining 18 h post-insemination (hpi). Two Day-7 blastocysts were transferred to each recipient ewe (n = 9; 3 per group) and pregnancies diagnosed by ultrasound on Day 57 of gestation. Data were analyzed by chi-square test. Oocyte maturation was 83.9% (73/87), and monospermic fertilization did not differ for Unsorted (22/32; 68.7%), X- (6/14; 42.8%), and Y-sperm groups (15/27; 55.6%). Polyspermic fertilization was 9.4% (3/32) and 7.4% (2/27) for the Unsorted and Y groups. Cleavage was reduced with X- and Y-sperm compared with Unsorted, but blastocyst formation (from cleaved oocytes) did not differ (Table 1). There were three (100%), zero (0%), and one (33.3%) pregnancies from Unsorted, X- and Y-embryos, respectively, all of which survived to birth, demonstrating that juvenile breeding can be successfully combined with sperm sexing. Table 1 Cleavage and blastocyst formation after IVF with Unsorted, X- or Y-sperm. Values in parenthesis are percentages

A range of tachykinins including substance P were studied for their ability to contract the guinea-pig ileum longitudinal muscle preparation on brief exposure (20 s) to the peptides, and their ability to evoke the release of [3H]-acetylcholine (ACh) from previously labelled stores within the myenteric plexus. With respect to their immediate spasmogenic activity, none of the peptides differed greatly in potency from substance P. Atropine did not modify the response to the tachykinins suggesting that the release of ACh does not contribute to the contraction resulting from brief exposure to the peptides. In the release studies, all tachykinins used produced a dose-related, calcium-dependent release of [3H]-ACh but the differences in potency were much greater. Eledoisin was the most potent and its evoked release of ACh was unaffected by hyoscine, hexamethonium, guanethidine and naloxone suggesting the release is not mediated via, or modulated by, opiate or autonomic neuronal influences. The two orders of tachykinin potency found suggest that the two processes, initial contraction and ACh release, may be principally mediated via two distinct subclasses of substance P receptor designated SP-P and SP-E respectively.

Prostaglandin (PG) A, E and F compounds applied serosally inhibited net fluid absorption and increased intraluminal pressure in guinea-pig isolated gallbladder. The inhibition was dose-dependent for a given animal body weight but sensitivity varied inversely with animal body weight; relative potencies were PGE2 greater than F2alpha greater than A1. In heavier animals the inhibition was preceded by an apparent increase in fluid absorption, due to fluid extrusion following muscle contraction. Net fluid secretion was observed at higher concentrations of PGE2 or F2alpha in lighter animals. Mucosally applied PGs less potently inhibited absorption, with relative potencies of PGE2 greater than A1 greater than F2beta greater than F2alpha. The spasmogenic effect was estimated by measuring intraluminal pressure (PGE2 greater than F2alpha greater than A1).

The potencies of three tachykinin antagonists [D-Pro4,D-Trp7,9,10]SP(4-11), [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP(1-11) and [D-Arg1,D-Trp7,9,Leu11]SP(1-11) (spantide) against eledoisin were examined in the guinea-pig ileum myenteric plexus, where a continuous superfusion system was employed to examine evoked release of [3H]-acetylcholine [( 3H]-ACh]); effects on mechanical activity of the preparations were also measured. Eledoisin was chosen as the standard tachykinin agonist since the rank order of potency observed in evoking release was eledoisin, kassinin, substance P, physalaemin; on this basis is may be presumed that an 'SP-E' type receptor was involved in the release process. The two undecapeptide antagonists both significantly reduced the response to eledoisin (10 nM) as assessed by both [3H]-ACh release and mechanical activity which under these conditions was largely dependent on ACh release, and the response levels could be restored by increasing the concentration of eledoisin to 100 nM. The pA2 values for the two antagonists were estimated as 5.3 for [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP(1-11) and 5.2 for [D-Arg1,D-Trp7,9,Leu11]SP(1-11). [D-Pro4,D-Trp7,9,10]SP(4-11) was markedly less potent with a pA2 value of less than 4.8. All three antagonists possessed considerable inherent stimulatory activity as measured both by [3H]-ACh release and mechanical activity, [D-Pro4,D-Trp7,9,10]SP(4-11) being the most active in this respect, a 10 microM concentration producing 50% of the response seen with 10 nM eledoisin. These findings are discussed both in relation to tachykinin receptor classifications and limitations in the use of such antagonists in the study of the role of tachykinins in neurotransmission.