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±115.5nmolL⁻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±156.5nmolL⁻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.
Summary
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.
The production of equine embryos by somatic cell nuclear transfer (SCNT) is limited by the availability of immature oocytes. The main source of oocytes for SCNT comes from ovaries obtained from ...slaughter houses. However, in some countries there is no horse abattoirs or their number is limited which precludes the use of SCNT for the production of equine cloned embryos. Ovum pick-up (OPU) is a reproductive assisted technique which is growing rapidly in the equine industry which allows obtaining oocytes from live mares. OPU is mainly used in combination to ICSI (intracytoplasmic sperm injection) for the production of in vitro embryos. Oocytes collected from live recipient mares by OPU could be an alternative for SCNT in countries in which there are limited number of equine slaughter houses. However, it is unknown the effect of the oocytes’ source on the development of equine cloned embryos. The aim of this study was to compare the development of equine SCNT embryos produced using oocytes either retrieved by OPU or recovered from abattoir-sourced ovaries. For the study, a total of 1128 oocytes were used, of which 663 were from abattoir-sourced ovaries and 495 were from live mares programmed for OPU. The harvested ovaries were transported to the lab within 1 h for processing. The methods used for in vitro maturation (IVM), SCNT andembryo culture in vitro were identical for both sources of oocytes. The rates of maturation, cell fusion, cleavage and blastocyst formation at Day 7 were evaluated. Transferrable grade blastocysts were transferred to recipient mares 4 to 5 days after ovulation and pregnancy was diagnosed at Days 14 and 42 by ultrasound. The maturation rate of OPU-derived oocytes was lower than that of abattoir-derived oocytes (50.3 ± 2.7% vs. 61.9 ± 3.4%; P < 0.05). However, the rates of cell fusion (90.7 ± 2.6% vs. 81.9 ± 5.2%), cleavage (68.8 ± 3.9% vs. 61.9 ± 5.0%) and blastocyst formation (34.3 ± 2.8% vs. 25.4 ± 2.1%) were greater (P < 0.05) for OPU-derived embryos compared with abattoir-derived embryos. While similar proportions of OPU- and abattoir-derived blastocysts initiated pregnancy at Day 14, a greater proportion of OPU-derived blastocysts developed to Day 42 of gestation (13 of 50 embryos) compared with abattoir-derived blastocysts (3 of 27 embryos; P < 0.05). The results show that abattoir-sourced oocytes subjected to IVM have the potential to support the development of SCNT embryos that can initiate pregnancy. However, given the observed difference in subsequent in vivo development, the use of OPU-derived oocytes for equine SCNT embryo production is recommended. Further research is needed to improve the IVM of abattoir-sourced oocytes
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.
KASCADE and its extension array of KASCADE-Grande were devoted to measure individual air showers of cosmic rays in the primary energy range of 100 TeV to 1 EeV. The experiment has substantially ...contributed to investigate the energy spectrum and mass composition of cosmic rays in the transition region from galactic to extragalactic origin of cosmic rays as well as to quantify the characteristics of hadronic interaction models in the air shower development through validity tests using the multi-detector information from KASCADE-Grande. Although the data accumulation was completed in 2013, data analysis is still continuing. Recently, we investigated the reliability of the new hadronic interactions models of the SIBYLL version 2.3d only with the energy spectra from the KASCADE-Grande data. The evolution of the muon content of high energy air showers in the atmosphere is studied as well, using EPOS-LHC, SIBYLL 2.3, QGSJET-II-04 and SIBYLL 2.3c. In this talk, recent results from KASCADE-Grande and the update of the KASCADE Cosmic Ray Data Centre (KCDC) will be discussed.
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 β-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.
Niacin deficiency has recently been associated with congenital malformations in humans and embryonic death and resorption in mice (Shi et al. New England Journal of Medicine. 2017; 377(6):544-552). ...Mares suffer from high rates of early embryonic loss (Newcombe. Equine Veterinary Education. 12(2):88-101), the aetiology of which remains unknown. Increasing the levels of nicotinamide adenine dinucleotide (NAD+) through supplementing NAD+ precursors has resulted in an improvement in oocyte quality in mice (Bertoldo et al. Cell Rep. 2020; 30: 1670-1681). However, little is known regarding the requirement of niacin and NAD+ in reproductive processes in the mare. The aim of this study was to determine the effect of oral nicotinic acid (NA) supplements, a form of niacin, on the composition of nicotinamide adenine dinucleotide (NAD+) metabolites in the blood and follicular fluid of mares. Vehicle alone or NA (3 g per os) was administered to 7 mares over a minimum of 3 consecutive days during the oestrous cycle. Blood samples were collected immediately prior tosupplemental feeding and at follicular fluid aspiration. Follicular fluid was collected from the dominant follicle through transvaginal ultrasound guided aspiration. Blood and follicular fluid samples were processed and analysed by mass spectrometry. The concentration of nicotinamide mononucleotide (NMN) in the follicular fluid of NA-fed mares was greater than that in the corresponding plasma (15.0 ± 6.5 vs. 3.3 ± 0.9 ng/µL; p < 0.05) and in the follicular fluid of vehicle-fed mares (1.5 ± 0.5 ng/µL; p < 0.001). Meanwhile, the concentration of NA, nicotinamide (NAM) and nicotinuric acid (NUR) tended to be greater in the follicular fluid of NA supplemented mares (1043.9 ± 953.5, 1641.7 ± 256.1 and 5.5 ± 5.1 ng/µL) than in the corresponding plasma (70.8 ± 15.4, 875.6 ± 361.1 and 0.6 ± 0.2, respectively; p < 0.1). The results show that NA supplementation increased the bioavailability of NAD+ precursors in the follicular fluid of the dominant follicle, which is proposed to better promote the maturation of good quality oocytes, especially in older mares.
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.
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.
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 15×106 motile, frozen-thawed sperm were inseminated laparoscopically at 50, 54, 58, 62, and 66h after progestagen withdrawal. An additional treatment dose of 0.5×106 S or NS sperm was inseminated at the 58h time point (n=60). Pregnancy was diagnosed by ultrasound at 60–62 d gestation. Both 1×106 and 15×106 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 15×106 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 0.5×106 motile sperm at 58h after PR (15±3.6% vs. 14±3.3%), nor with 15×106 motile sperm at all insemination times (49±6.3% vs. 49±6.3%). However, fertility was higher for S than NS sperm at the 1×106 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.
