Multiple ovulation and embryo transfer in sheep: Effects of embryo developmental stage and quality on viability in vivo under farm conditions.

Multiple ovulation and embryo transfer (MOET) technologies are integral to genetic improvement programs in the sheep industries. Despite the protocols being well established, previous findings regarding the effects of embryo properties on MOET success remain contradictory. The objective of this study was to determine the effects of embryo developmental stage and quality on embryo viability following transfer to recipient ewes. Data including details of 377 embryos collected from 45 Merino donor ewes were obtained from MOET trials conducted on three separate farms on day 6 after laparoscopic artificial insemination (AI). A total of 270 embryos were classified as being of transferrable grade (grade 1: n = 233; grade 2: n = 37). One or two transferrable grade embryos were transferred to each of 256 synchronised recipient ewes and pregnancy diagnosis was performed on day 36 after embryo transfer. Embryos at the hatched blastocyst stage tended to have greater viability in vivo compared to embryos at the late morula stage (59.0 ± 10.6% vs. 36.2 ± 9.7%; P = 0.083). The viability of grade 1 embryos was greater than that of grade 2 embryos (53.6 ± 7.8% vs. 35.9 ± 10.2%; P < 0.05). The results suggest that the success of the MOET trials was influenced by the transfer of embryos at the late morula stage, almost half of which were classified as grade 2 embryos. These findings highlight the importance of following strict embryo quality grading criteria to inform the most economical management of recipient ewes and maximize pregnancy outcomes.

Supplementation of culture medium with L-carnitine improves the development and cryotolerance of in vitro-produced porcine embryos.

Porcine oocytes and embryos contain substantial amounts of lipid, with little known regarding its metabolic role during development. This study investigated the role of lipid metabolism and the interaction between carbohydrate and lipid substrates in porcine embryos. Following in vitro fertilisation, presumptive zygotes were transferred to culture medium supplemented with L-carnitine, a co-factor required for the metabolism of fatty acids. In porcine zygote medium-3 (PZM-3), which contains pyruvate and lactate, 3mM L-carnitine was the only dose that improved cleavage rates compared with the control. In the absence of carbohydrates, all doses of L-carnitine from 1.5 to 12mM increased cleavage rates compared with the control. Culture in a PZM-3-based sequential media system (Days 0-3: pyruvate and lactate; Days 4-7: glucose) significantly increased blastocyst cell numbers compared with culture in standard PZM-3. Supplementing PZM-3 with 3mM L-carnitine produced blastocysts with cell numbers equivalent to those obtained in the sequential media system. After vitrification, the post-warming survival rates of blastocysts obtained in media supplemented with 3mM L-carnitine were significantly greater than those of blastocysts obtained in standard PZM-3. In conclusion, L-carnitine supplementation improved embryo development when the medium contained pyruvate and lactate or was lacking carbohydrates completely, indicating a role for fatty-acid metabolism when the embryo's requirements for carbohydrates are not adequately met.

Field fertility of liquid stored and cryopreserved flow cytometrically sex-sorted stallion sperm.

REASONS FOR PERFORMING STUDY: The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. OBJECTIVES: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. STUDY DESIGN: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. METHODS: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted to 25 or 111 × 106 sperm/ml (-SP25 and -SP111). Sperm were stored for 18 h at 10 to 15°C and -SP25 and +SP25 treatments were centrifuged and resuspended to 111 × 106 sperm/ml. Sperm were incubated under H33342 staining conditions and motility, viability and acrosome integrity assessed. Semen was collected from stallions (n = 4), liquid stored at 10-15°C for up to 5 h and sperm either cryopreserved directly, sex-sorted and cryopreserved, or sex-sorted and returned to liquid storage until insemination. Low-dose hysteroscopic insemination was performed in 23 mares randomly allocated to the semen preparation group and pregnancy determined following embryo flushing on Day 9 after ovulation, or via transrectal ultrasonography on Day 14 after ovulation. RESULTS: Skimmed milk was superior to I-BSA in maintaining motility, viability and acrosome integrity. Seminal plasma removal did not affect the parameters measured at the concentrations examined. Conception rates did not differ significantly between the groups, although a high incidence of pregnancy loss was observed in both the cryopreserved groups. CONCLUSIONS: While the conception rates achieved are among the highest yet reported for sex-sorted, cryopreserved stallion sperm, the high incidence of pregnancy loss suggests that the development of the resulting embryos was significantly impaired by the sperm processing treatments.

Proteomic characterization and cross species comparison of mammalian seminal plasma.

Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. BIOLOGICAL SIGNIFICANCE: This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm.

Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index [DFI], mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona binding ability of cryopreserved stallion sperm, and reduced DNA fragmentation in sex-sorted, cryopreserved stallion sperm.

Dimethyl formamide improves the postthaw characteristics of sex-sorted and nonsorted stallion sperm.

Cryopreserved, sex-sorted stallion sperm has been shown to have poor fertility. During this study, the effects of cryoprotectant (glycerol [GLY] and dimethyl formamide [DMF]), cryoprotectant equilibration time (0, 30, 60, 90, or 120 minutes), and cryoprotectant concentration (2%, 3%, or 4% vol/vol) on stored sex-sorted and stored nonsorted stallion sperm were evaluated. Total motility, viability, and DNA integrity (determined using sperm chromatin structure assay) of sperm were assessed after thawing. Equilibration for 90 minutes improved total motility (33.8%) compared with 0 (28.5%) or 120 minutes (29.8%; P < 0.05), though viability was higher after 120 minutes (33.1%) compared with 0 (30.5%) or 30 minutes (31.0%; P < 0.01). The viability of nonsorted sperm decreased as cryoprotectant concentration increased (P < 0.001), and total motility of nonsorted sperm was higher when DMF alone was used (15.8%, 16.6%, and 24.0% for GLY, GLY and DMF, and DMF respectively; P < 0.001). Sex sorting was detrimental to the postthaw quality of sperm; at 45 minutes after thawing, total motility of nonsorted sperm was higher than that of sex-sorted sperm (37.4% vs. 5.6%; P < 0.001), the viability of sex-sorted sperm was lower than that of nonsorted sperm (12.4% vs. 30.0%; P < 0.001, averaged over postthaw time), and sex-sorted sperm had higher detectable DNA fragmentation index (DFI) (63.6% vs. 11.3%, P < 0.001) and mean DFI (285.1 vs. 211.3, P < 0.001) than nonsorted sperm. The viability of sex-sorted sperm was improved by GLY and DMF or DMF compared with GLY (22.6%, 25.3%, and 19.3%, respectively; P < 0.05), and the DNA integrity of sex-sorted sperm was improved by the use of DMF compared with GLY (detectable DFI, 60.2 vs. 66.8, P < 0.05; and mean DFI, 280.9 vs. 289.2, P < 0.05, respectively). In conclusion, postthaw characteristics of stored sex-sorted and stored nonsorted stallion sperm were improved by the use of DMF as a cryoprotectant, though the parameters to benefit differed between sorted and nonsorted sperm.

Seasonal variation in the ovarian function of sows.

The modern domestic sow exhibits a period of impaired reproductive performance known as seasonal infertility during the late summer and early autumn months. A reduction in farrowing rate due to pregnancy loss is the most economically significant manifestation of this phenomenon. Presently, little is known of the aetiology of seasonal pregnancy loss in the pig. Recent findings represent a major advancement in the understanding of sow reproductive physiology and implicate poor oocyte developmental competence as a contributing factor to pregnancy loss during the seasonal infertility period. It has also been demonstrated that ovarian activity is depressed during the seasonal infertility period. The reduction in oocyte quality is associated with decreased levels of progesterone in follicular fluid during final oocyte maturation in vivo. The recent identification of sow-specific risk factors, such as parity for late pregnancy loss, should improve breeding herd efficiency by allowing producers to tailor management interventions and/or culling protocols that target animals identified as having a greater risk of late pregnancy loss during the seasonal infertility period.

Quantitative mRNA expression in ovine blastocysts produced from X- and Y-chromosome bearing sperm, both in vitro and in vivo.

Artificial insemination (AI) of sex-sorted sperm results in decreased fertility, compared with non-sorted sperm, in most species. However, this has not been the case in sheep, where the low-dose AI of sex-sorted ram sperm produced similar, if not superior, fertility to non-sorted controls. The aim of the present study was to determine the impact of sex-sorting technology on ovine embryo gene expression following embryo production in vivo and in vitro. After semen collection, ejaculates were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen. Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched sperm, or non-sorted control sperm, and collected by uterine flushing on Day 6 after AI. Embryos were produced in vitro using the same sperm treatments and cultured in vitro for 6 d. The relative abundance of selected gene transcripts was measured in high-grade blastocysts, defined by morphological assessment, using RT-qPCR. The mRNA expression of DNMT3A and SUV39H1 was upregulated in embryos cultured in vitro, compared to those cultured in vivo (DNMT3A: 3.61 ± 1.08 vs 1.99 ± 0.15; SUV39H1: 1.88 ± 0.11 vs 0.88 ± 0.07; mean ± SEM; P < 0.05). Both G6PD and SLC2A3 transcripts were reduced in embryos produced from sex-sorted sperm, in vivo (SLC2A3: 0.23 ± 0.03 vs 0.64 ± 0.10; G6PD: 0.32 ± 0.04 vs 1.01 ± 0.16; P < 0.05). The expression of DNMT3A was up-regulated in male (3.85 ± 0.31), compared to female embryos (2.34 ± 0.15; P < 0.05). This study contributes to the growing body of evidence citing aberrant patterns of gene expression resulting from in vitro culture. Whereas the process of sex-sorting altered the expression of several of the genes examined, no effect on embryo development was detected.

Seasonal effects on oocyte developmental competence in sows experiencing pregnancy loss.

Recently, oocyte quality in sows culled for reasons unrelated to fertility was found to decline during the period of seasonal infertility. Wean-to-service interval (WSI) has also been associated with pregnancy loss in sows mated during the period of seasonal infertility. The aims of this study were to determine whether WSI and season are associated with changes in oocyte developmental competence in sows experiencing early (before Day 35 of gestation) and late (after Day 35 of gestation) pregnancy loss. Ovaries were collected in pairs from sows sourced from commercial piggeries that were culled for reasons related to infertility after being mated in summer and winter/spring. Sows were grouped according to their pregnancy loss type, their previous WSI and the presence or absence of corpora lutea (CL) on their ovaries. Oocyte developmental competence was assessed following in vitro maturation, artificial activation and parthenote development in vitro. In sows culled for early-pregnancy loss, there was a greater number of CL present on ovaries collected in spring compared to those collected in summer (11.57±3.3 vs. 9.26±0.99; P<0.05). Also, the proportion of oocytes developing to the blastocyst stage was greater in summer than in spring (55.9±5.2% vs. 31.2±6.4%; P<0.05). In sows culled for late-pregnancy loss, a greater proportion of oocytes developed to the blastocyst stage in winter compared with late-spring (64.3±7.0% vs. 34.1±6.6%; P<0.05). In addition, the blastocyst formation rate of oocytes was lower in sows that displayed a WSI≤6 days than in sows that displayed a WSI>6 days (37.8±7.3% vs. 62.2±6.9%; P<0.05). The results of the present study indicate that sows culled for pregnancy loss exhibit seasonal changes in oocyte developmental competence. The mechanism which causes WSI to be prolonged does not appear to result in reduced oocyte developmental competence. While poor oocyte quality and the mechanism that increases WSI may contribute to pregnancy loss during the seasonal infertility period, the findings suggest that these factors are not the main drivers of early and late pregnancy loss throughout the year.

Follicular progesterone levels decrease during the period of seasonal infertility in sows.

Impaired reproductive performance exhibited by the domestic sow during the late summer and early autumn months is referred to as seasonal infertility. This study was carried out to determine whether there are changes in ovarian morphology and follicular steroidogenesis associated with season, which may be associated with seasonal infertility. Ovaries were collected in pairs from sows sourced from two farms and slaughtered 4 days after weaning during winter and summer. The mean progesterone concentration in follicular fluid (FF) collected from small follicles was lower in summer (701.3 ± 115.54 nm) compared with winter (1235.55 ± 164.47 nm; p<0.001). The mean progesterone concentration in the FF of large follicles was also lower in summer (1469.2 ± 156.51 nm) compared with winter (2470.9 ± 169.13 nm; p<0.001). The number of large surface antral follicles (5-8 mm in diameter) on the ovaries recovered from Farm A sows was higher during summer (17.76 ± 0.56) than in winter (15.38 ± 0.54; p<0.05). Similarly, the number of small follicles (3-4 mm in diameter) on Farm A sow ovaries was higher in summer (8.46 ± 0.66) than in winter (4.63 ± 0.53; p<0.001). In contrast, the number of small follicles on the surface of ovaries recovered from Farm B sows was higher during winter (10.17 ± 1.50) than in summer (6.45 ± 1.00; p<0.01). The number of pre-ovulatory follicles (>8 mm in diameter) was also higher in winter (1.23 ± 1.68) when compared to summer (0.51 ± 0.3; p<0.001) on the ovaries of sows from Farm B. The results suggest that there are seasonal differences in follicular steroidogenesis and ovarian dynamics. These findings add support to the theory that altered follicular steroidogenesis and ovarian morphology may possibly be the mechanism behind reduced reproductive performance during the period of seasonal infertility in sows.

Use of a defined diluent increases the sex-sorting efficiency of stallion sperm.

The low efficiency of flow cytometric sex-sorting of stallion sperm has been attributed to the use of an opaque skim milk-based diluent during Hoechst 33342 (H33342) staining. Three experiments were conducted to formulate an optically clear stallion semen diluent for use during H33342 staining, and to determine whether a clear diluent improved resolution during sorting. For Experiment 1, sperm were incubated at 34 °C in each of five diluents containing either no protein, skim milk, 0.25% Cohn's Fraction V BSA, 0.5% BSA, or 1% BSA, following an 18 h storage (15 °C) period, or shortly after collection. Sperm incubated in both skim milk and 1% BSA-supplemented diluents had equivalent total (47 and 49.5%, respectively) and progressive (4.73 and 5.67%, respectively) sperm motilities after 45 min, and comparable acrosome integrity (65.9 and 67.9%, respectively). For Experiment 2, the protein source was optimised by comparing the characteristics of sperm stored and incubated in five diluents supplemented with skim milk, BSA, fatty acid and endotoxin free BSA (I-BSA), KnockOut™ Serum Replacement, and ß-lactoglobulin, respectively. The I-BSA diluent was superior to skim milk for motility maintenance during incubation (74.0 vs 63.7%). The effect of diluent on sorting was investigated in Experiment 3 using a range of H33342 concentrations and incubation durations. The clear (1% BSA) diluent improved the split ratio compared with the opaque (skim milk) diluent (0.17 vs 0.08), with an optimum staining time of 45 min using 0.09 mM H33342. In conclusion, a diluent containing 1% fatty acid free, low endotoxin BSA in lieu of skim milk improved the sorting efficiency and motility characteristics of stallion sperm after storage for 18 h.

Oocyte developmental competence is reduced in sows during the seasonal infertility period.

The modern domestic sow exhibits a period of impaired reproductive performance during the late summer and early autumn months, known as 'seasonal infertility'. A reduction in farrowing rate due to pregnancy loss is the most economically important manifestation of seasonal infertility. The aim of the present study was to determine whether there are changes in oocyte developmental competence associated with season. Ovaries were collected in pairs from sows sourced from commercial piggeries and slaughtered 4 days after weaning during winter and summer-autumn. Following oocyte IVM and parthenogenetic activation, the ability of oocytes from large follicles to form blastocysts was greater in winter (54.94 ± 6.11%) than in summer (21.09 ± 5.59%). During winter, the proportion of oocytes developing to the blastocyst stage from large follicles was significantly higher (54.94 ± 6.11%) than those oocytes from small follicles (23.17 ± 6.02%). There was no effect of season on the proportion of oocytes developing to the blastocyst stage from small follicles. There was no effect of follicle size on blastocyst formation from those oocytes recovered during summer. Blastocysts derived from small follicles during summer had the lowest number of cells (24.25 ± 1.48) compared with blastocysts derived from large follicles during winter (37.5 ± 1.3; P < 0.05). The mean progesterone concentration in follicular fluid collected from small follicles was greater in winter than summer (1235.55 ± 164.47 v. 701.3 ± 115.5 nmol L(-1), respectively; P < 0.001). The mean progesterone concentration in the follicular fluid of large follicles was also greater in winter than in summer (2470.9 ± 169.1 v. 1469.2 ± 156.5 nmol L(-1), respectively; P < 0.001). Regression analysis revealed a positive correlation between progesterone concentration and oocyte developmental competence. The results indicate that porcine oocytes fail to reach their full developmental potential during the period of seasonal infertility, suggesting that the pregnancy losses observed at this time of year may be due to reduced oocyte developmental competence.

The effect of sperm and cryoprotectant concentration on the freezing success of sex sorted ram sperm for in vitro fertilization.

The current method used to sex-sort ram sperm resulted in a dilute end product. The obligatory removal of cryopreservation medium during preparation of sperm IVF further reduced sperm number. This study aimed to increase the number of viable, sex-sorted sperm available for IVF by increasing their pre-freeze concentration and assessing the cryodiluent concentration used to accommodate this change. In Experiment 1, semen was collected from Merino rams (n = 3), sex-sorted, and then frozen at concentrations of 20, 40, or 80 x 10(6) sperm/mL in three forms of tris-citrate-glucose cryodiluent containing 5% (L-Cryo), 6% (M-Cryo), and 8% (H-Cryo) (v/v) glycerol. Motility, plasma membrane and acrosome integrity, and mitochondrial activity were assessed at 0, 2, 4, and 6 h post thaw. In Experiment 2, cleavage and blastocyst development rates were compared between non-sorted and sex-sorted sperm frozen at the aforementioned concentrations (in the cryodiluent most effective in Experiment 1). In Experiment 1, total motility between 0 and 6 h was similar for all sperm concentrations when frozen using L-Cryo. Mitochondrial activity was elevated in samples frozen in L-Cryo and M-Cryo at 0 h compared to those preserved in H-Cryo for all concentrations (P < 0.05). In Experiment 2, sex-sorted sperm with a higher pre-freeze concentration yielded a higher sperm concentration after preparation for IVF (8.57 +/- 1.22 sperm/mL), compared to the lowest group (2.96 +/- 0.18 sperm/mL; P < 0.05). There were no significant differences between non-sorted and sex-sorted sperm for rates of embryo cleavage or development. Therefore, sex-sorted sperm was effectively cryopreserved at a higher concentration than conventionally practiced. Although this yielded a higher sperm concentration for IVF, reduced insemination volume, and increased the number of potentially fertile gametes from which to select, fertilisation rate was not significantly improved.

Identification of sow-specific risk factors for late pregnancy loss during the seasonal infertility period in pigs.

Reduced farrowing rates due to late pregnancy loss (LPL) is a manifestation of seasonal infertility in pigs. This study was undertaken to determine sow- and gilt-specific risk factors leading to LPL during the seasonal infertility period (January to April) in Australia. Age at first service was considered to be a major gilt-specific risk factor, whereas sow-specific factors considered included parity, prior wean-to-service interval, prior lactation length, and number of piglets weaned in the lactation period immediately preceding the mating/pregnancy event under scrutiny. Logistic regression analysis of these factors was undertaken on 13,213 animals from three farms (Farms A, B, and C). Age at first service for gilts had an effect on LPL (P<0.05) on Farm C when compared with that for Farms A and B, with those mated at approximately 220 d having the lowest rate of LPL. For older sows, parity was a factor on Farms A and C (P<0.001), with the proportion of sows with LPL increasing with increasing parity. When the data from each farm were combined and analyzed, there was a significant farm by WSI interaction, with animals from Farm C being most at-risk for LPL. Sows with shorter lactation periods (P<0.05) and smaller litters (P<0.05) at the previous lactation had a greater chance of LPL on all farms. Under the conditions of this study, we were able to identify risk factors for LPL that producers can manipulate during the seasonal infertility period to improve breeding herd productivity.

The effect of insemination time and sperm dose on pregnancy rate using sex-sorted ram sperm.

The objective was to determine the optimum timing of insemination and minimum effective dose rate of sex-sorted ram sperm. Semen from three Merino rams was sorted into high purity X- and Y-chromosome bearing sperm populations. Ovulation was controlled in 732 Merino ewes using PMSG at progestagen pessary removal and GnRH 36h later. Sorted (S) and non-sorted (NS) doses of 1 or 15x10(6) motile, frozen-thawed sperm were inseminated laparoscopically at 50, 54, 58, 62, and 66h after progestagen withdrawal. An additional treatment dose of 0.5x10(6) S or NS sperm was inseminated at the 58h time point (n=60). Pregnancy was diagnosed by ultrasound at 60-62 d gestation. Both 1x10(6) and 15x10(6) sperm achieved similar pregnancy rates, regardless of sperm type, at 58h (S1: 46+/-9.4%; S15: 43+/-9.3%; NS1: 41+/-9.2%; NS15: 49+/-9.4%). However, pregnancy rates were lower (P<0.05) for doses of 1 than 15x10(6) sperm inseminated at 50 (15+/-6.3% vs. 36+/-9.1%), 54 (14+/-4.4% vs. 55+/-7.3%), 62 (33+/-6.9% vs. 54+/-7.3%), and 66h (29+/-8.6% vs. 56+/-9.5%). There was no difference between S and NS sperm for inseminations with 0.5x10(6) motile sperm at 58h after PR (15+/-3.6% vs. 14+/-3.3%), nor with 15x10(6) motile sperm at all insemination times (49+/-6.3% vs. 49+/-6.3%). However, fertility was higher for S than NS sperm at the 1x10(6) dose level (37+/-6.1% and 16+/-4.0%). More than 90% of lambs born were of the predicted sex. We hypothesise that the sorting process selects a homogeneous, fertile sub-population of sperm, removing those that are dead, damaged and morphologically abnormal.

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus).

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig.

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly (P<0.01) larger than NCL ovaries (width: 22.3+/-0.9 mm versus 15.9+/-0.4 mm, length: 33.2+/-1 mm versus 24.1+/-0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1+/-1.8 mm versus 46.8+/-2.2 mm), they had a similar number of follicles 3-8mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5+/-0.1 mm versus 3.3+/-0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159+/-1.3 microm versus 146.1+/-1.5 microm, zona free: 124.7+/-1.8 microm versus 113.1+/-1.6 microm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6+/-3.2% versus 92.4+/-3.2%), cleavage (88.4+/-11% versus 90.7+/-2.6%) and blastocyst formation (21.0+/-3.7% versus 23.7+/-5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.

Effect of DNA concentration on transgenesis rates in mice and pigs.

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10 ng/microl, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5 ng/microl and then decreased at 10 ng/microl for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10 ng/microl to maximise integration and efficiency rates in pigs.

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts.

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred.

Activation of in vivo- and in vitro-derived porcine oocytes by using multiple electrical pulses.

The current protocols used to activate pig nuclear transfer embryos are less efficient than those used for other species. To address this problem, the effect of multiple sets of electrical pulses on the parthenogenetic development of in vivo- and in vitro-derived porcine oocytes was examined. Each set of pulses consisted of two 1.5 kV cm(-1) DC pulses of 60 micros duration each, administered 1 s apart. For in vivo-derived oocytes, application of a second set of pulses 30 min after the first set increased the proportion of oocytes that developed to the blastocyst stage compared with a single treatment (51 v. 34%). Application of a third set of pulses 30 min after the second set reduced the rate of blastocyst formation compared with two sets of pulses. In contrast, the rate of blastocyst formation was greater with one set of pulses compared with two sets for in vitro matured oocytes (31 v. 16%). Additional sets of electrical pulses did not affect the number of cells in blastocysts obtained from either group of oocytes compared with a single treatment. In summary, the study demonstrates that the application of a second set of activating pulses 30 min after the first set is beneficial to in vivo-derived oocytes, but detrimental to in vitro matured oocytes, in terms of their ability to develop parthenogenetically to the blastocyst stage.