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.
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.
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.
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.
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•Cross species comparison of the seminal plasma proteomes of seven mammalian species•The similarity of seminal proteomes can be related to the phylogeny of the species.•Beta nerve growth factor is the major protein in alpaca and camel seminal plasma.•Bodhesins and zinc alpha glycoprotein are a signature of small ruminant seminal plasma proteomes.•Nucleobindin-1 and RSVP14 proteins are common to the seven species.
We report the observation of a steepening in the cosmic ray energy spectrum of heavy primary particles at about 8×10(16) eV. This structure is also seen in the all-particle energy spectrum, but is ...less significant. Whereas the "knee" of the cosmic ray spectrum at 3-5×10(15) eV was assigned to light primary masses by the KASCADE experiment, the new structure found by the KASCADE-Grande experiment is caused by heavy primaries. The result is obtained by independent measurements of the charged particle and muon components of the secondary particles of extensive air showers in the primary energy range of 10(16) to 10(18) eV. The data are analyzed on a single-event basis taking into account also the correlation of the two observables.
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.
We present the results of the search for large-scale anisotropies in the arrival directions of cosmic rays performed with the KASCADE-Grande experiment at energies higher than eV. To eliminate ...spurious anisotropies due to atmospheric or instrumental effects we apply the east-west method. We show, using the solar time distribution of the number of counts, that this technique allow us to remove correctly the count variations not associated to real anisotropies. By applying the east-west method we obtain the distribution of number of counts in intervals of 20 minutes of sidereal time. This distribution is then analyzed by searching for a dipole component; the significance of the amplitude of the first harmonic is 3.5 , therefore, we derive its upper limit. The phase of the first harmonic is determined with an error of a few hours and is in agreement with the measurements obtained in the 1014 < E < 2 × 1015 eV energy range by the EAS-TOP, IceCube, and IceTop experiments. This supports the hypothesis of a change of the phase of the first harmonic at energies greater than ∼2 × 1014 eV.
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.
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 extreme cryo-sensitivity of porcine oocytes and embryos is attributed to their endemically high content of cytoplasmic lipid droplets. In attempts to improve the cryo-tolerance of porcine ...embryos, various strategies have been used to reduce the amount of lipid droplets present in the cytoplasm before vitrification. Recently, the cryo-tolerance of bovine oocytes vitrified at the metaphase II stage was improved by supplementing in vitro maturation (IVM) medium with l-carnitine (LC), a stimulator of lipid metabolism (Chakitisakul et al. 2013 Theriogenology 79, 590–598). The objective of this study was to determine the effect of supplementing IVM medium with LC on the post-thaw development of porcine embryos vitrified at the pronuclear stage. Oocytes recovered from the ovaries of prepubertal gilts were matured in modified porcine oocyte medium supplemented with 0 (control) or 12mM LC during the final 22h of IVM. Following IVF, presumptive zygotes were cultured in porcine zygote medium-3. At the pronuclear stage, cohorts of embryos from each group were either vitrified using a solid surface vitrification procedure (Somfai et al. 2009 Biol. Reprod. 80, 42–49) or cultured for 7 d without being vitrified. Vitrified zygotes were subsequently warmed and cultured for 7 d. The rates of cleavage, blastocyst formation, and hatching were recorded, and all blastocysts were stained to determine the total cell numbers. Three replicates were performed. Proportional data were arcsine transformed and subjected to ANOVA, and cell number data were analysed by t-test. The post-thaw survival rates of the embryos that were vitrified did not differ between the groups (control: 95.7%; LC: 97.1%; P>0.05). There were no significant effects of LC treatment or vitrification on the rates of cleavage, blastocyst formation, and hatching (Table 1). Vitrified embryos derived from LC-treated oocytes produced blastocysts with fewer cells than vitrified embryos derived from untreated oocytes (Table 1; P<0.05). In contrast to previous findings in other species, the results indicate that supplementing IVM medium with LC did not enhance the post-thaw development of porcine embryos vitrified at the pronuclear stage. Table 1.Effect of l-carnitine (LC) treatment and vitrification on porcine embryo development C. Grupen was supported by an OECD Fellowship.
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.
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