Analysis of bovine blastocysts indicates ovarian stimulation does not induce chromosome errors, nor discordance between inner-cell mass and trophectoderm lineages.

Contemporary systems for oocyte retrieval and culture of both cattle and human embryos are suboptimal with respect to pregnancy outcomes following transfer. In humans, chromosome abnormalities are the leading cause of early pregnancy loss in assisted reproduction. Consequently, pre-implantation genetic testing for aneuploidy (PGT-A) is widespread and there is considerable interest in its application to identify suitable cattle IVP embryos for transfer. Here we report on the nature and extent of chromosomal abnormalities following transvaginal follicular aspiration (OPU) and IVP in cattle. Nine sexually mature Holstein heifers underwent nine sequential cycles of OPU-IVP (six non-stimulated and three stimulated cycles), generating 459 blastocysts from 783 oocytes. We adopted a SNP-array approach normally employed in genomic evaluations but reanalysed (Turner et al., 2019; Theriogenology125: 249) to detect levels of meiotic aneuploidy. Specifically, we asked whether ovarian stimulation increased the level of aneuploidy in either trophectoderm (TE) or inner-cell mass (ICM) lineages of blastocysts generated from OPU-IVP cycles. The proportion of Day 8 blastocysts of inseminated was greater (P < 0.001) for stimulated than non-stimulated cycles (0.712 ± 0.0288 vs. 0.466 ± 0.0360), but the overall proportion aneuploidy was similar for both groups (0.241 ± 0.0231). Most abnormalities consisted of meiotic trisomies. Twenty in vivo derived blastocysts recovered from the same donors were all euploid, thus indicating that 24 h of maturation is primarily responsible for aneuploidy induction. Chromosomal errors in OPU-IVP blastocysts decreased (P < 0.001) proportionately as stage/grade improved (from 0.373 for expanded Grade 2 to 0.128 for hatching Grade 1 blastocysts). Importantly, there was a high degree of concordance in the incidence of aneuploidy between TE and ICM lineages. Proportionately, 0.94 were "perfectly concordant" (i.e. identical result in both); 0.01 were imperfectly concordant (differing abnormalities detected); 0.05 were discordant; of which 0.03 detected a potentially lethal TE abnormality (false positives), leaving only 0.02 false negatives. These data support the use of TE biopsies for PGT-A in embryos undergoing genomic evaluation in cattle breeding. Finally, we report chromosome-specific errors and a high degree of variability in the incidence of aneuploidy between donors, suggesting a genetic contribution that merits further investigation.

Global transcriptomic response of bovine endometrium to blastocyst-stage embryos.

The aims of this study were (i) to investigate changes in the global transcriptome of bovine endometrial explants induced by exposure to blastocysts, (ii) to investigate if male and female blastocysts elicit a differential response in the endometrial transcriptome in vitro and (iii) to determine whether bovine endometrium responds to the presence of murine embryos. In Experiment 1, endometrial explants from the same uterus were cultured for 6 h with or without 20 in vitro-produced bovine blastocysts. In Experiment 2, endometrial explants were cultured with male or female bovine blastocysts produced in vitro by IVF either using sex-sorted semen or conventional unsorted semen followed by embryo sexing based on a biopsy. In Experiment 3, endometrial explants were cultured alone or in the presence of bovine blastocysts (n = 25) or murine blastocysts (n = 25). Following culture, explants were snap frozen and stored at -80°C until RNA extraction, qPCR or RNA-Seq. Culture with bovine blastocysts increased endometrial expression of 40 transcripts, all of which were interferon-tau induced. Culture with male or female bovine blastocysts increased transcript abundance of five classic interferon-stimulated genes (MX1, MX2, ISG15, OASY1, RSAD2) in explants; however, there was no difference in abundance of transcripts previously reported to be related to embryonic sex (IFNAR1, IFNAR2, CTGF, ARTN, SLC2A1, SLC2A5). Exposure to murine blastocysts did not elicit any detectable change in transcript abundance. These findings, coupled with our previous data, indicate that very local, interferon-tau-induced changes in endometrial gene expression occur in response to blastocysts; whether such changes play any role in subsequent pregnancy recognition remains to be established.

Blastocyst-induced changes in the bovine endometrial transcriptome.

The objectives of this study were (i) to determine whether blastocyst-induced responses in endometrial explants were detectable after 6- or 24-h co-culture in vitro; (ii) to test if direct contact is required between embryos and the endometrial surface in order to stimulate endometrial gene expression; (iii) to establish the number of blastocysts required to elicit a detectable endometrial response; (iv) to investigate if upregulation of five interferon-stimulated genes (ISGs) in the endometrium was specific to the blastocyst stage and (v) to test if alterations in endometrial gene expression can be induced by blastocyst-conditioned medium. Exposure of endometrial explants to Day 8 blastocysts in vitro for 6 or 24 h induced the expression of ISGs (MX1, MX2, OAS1, ISG15, RSAD2); expression of IFNAR1, IFNAR2, NFKB1, IL1B, STAT1, LGALS3BP, LGALS9, HPGD, PTGES, ITGB1, AKR1C4, AMD1 and AQP4 was not affected. Culture of explants in the presence of more than five blastocysts was sufficient to induce the effect, with maximum expression of ISGs occurring in the presence of 20 blastocysts. This effect was exclusive to blastocyst stage embryos; oocytes, 2-cell embryos or Day 5 morulae did not alter the relative abundance of any of the transcripts examined. Direct contact between blastocysts and the endometrial surface was not required in order to alter the abundance of these transcripts and blastocyst-conditioned medium alone was sufficient to stimulate a response. Results support the notion that local embryo-maternal interaction may occur as early as Day 8 of pregnancy in cattle.

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed-time artificial insemination protocol in Australian Bos indicus cattle.

Increasing use of fixed-time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN2 ) in a Tris-egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post-thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.

Development of a GnRH-PGF2α-progesterone-based synchronization protocol with eCG for inducing single and double ovulations in beef cattle.

Experiments were designed to investigate the effect of different doses and timing of an eCG treatment given during GnRH-based synchronization protocols on follicular dynamics and fertility in cattle. In Exp. 1, Angus heifers (n = 50) received a 7-d Ovsynch + progesterone protocol (on d 0, GnRH and progesterone insert were administered; on d 7, progesterone insert was removed and PGF2α was injected; and on d 9.5, GnRH was injected 56 h after progesterone removal) with eCG (0, 300, 500, 700, or 1,000 IU) administered on d 7. In Exp. 2, Angus cows (n = 27) received the same protocol as Exp. 1 and were assigned randomly to receive 0 or 400 IU eCG i.m. on d 2 or 7. In Exp. 3, Angus cows (n = 18) received a 6-d Ovsynch + progesterone protocol and were randomly assigned to receive 0 or 800 IU eCG on d 3 of the protocol (Exp. 3a). A pilot field trial was also performed using the same treatments in suckled Angus-cross cows (n = 72; Exp. 3b). In Exp. 4, beef heifers (n = 200) were assigned randomly to the same treatments as in Exp. 3, but the second GnRH was not given, with Holstein bulls introduced on d 6. In Exp. 5, Angus cows (n = 12) received the same treatment as in Exp. 3, but were not inseminated. Progesterone concentrations were assessed in plasma collected during the estrous cycle following synchronization. Ultrasonography was used to monitor ovarian dynamics and to diagnose pregnancy. In Exp. 1, the mean number of ovulations was affected (P < 0.02) by the dose of eCG and the stage of follicular development when administered. Treatment with eCG on d 2 tended (P < 0.08) to extend the interval from PGF2α to ovulation, but was not successful in inducing double ovulations. In contrast, eCG on d 3 increased (P < 0.01) the number of cows with double ovulation when administered i.m. and increased (P < 0.04) pregnancy rate in single ovulating heifers after bull breeding (68.0 vs. 53.1%). This treatment also elevated progesterone concentrations during the estrous cycle following synchronization. Thus, the mechanism by which administration of eCG on d 3 of the synchronization increased pregnancy rates may be through supporting development of a healthy follicle and subsequent corpus luteum capable of secreting increased concentrations of progesterone during early pregnancy. In conclusion, strategic administration of eCG during a synchronization protocol can be used to improve reproductive performance through increased pregnancy rates in single ovulating animals as well as the induction of twin ovulations for twinning.

Intravaginal progesterone devices in synchronization protocols for artificial insemination in beef heifers.

Two experiments were designed to investigate the administration of intravaginal progesterone in protocols for oestrus and ovulation synchronization in beef heifers. In Experiment 1, cyclic Black Angus heifers (n = 20) received an Ovsynch protocol and were randomly assigned to receive (CIDR-Ovsynch) or not (Ovsynch) a progesterone device between Days 0 and 7. Treatment with a controlled internal drug release (CIDR) device significantly increased the size of the dominant follicle prior to ovulation (12.8 ± 0.4 CIDR-Ovsynch vs 11.4 ± 0.4 Ovsynch) (p < 0.02). Plasma progesterone concentrations throughout the experiment were affected by the interaction between group and day effects (p < 0.004). In Experiment 2, cyclic Polled Hereford heifers (n = 382) were randomly assigned to one of the six treatment groups (3 × 2 factorial design) to receive a CIDR, a used bovine intravaginal device (DIB), or a medroxiprogesterone acetate (MAP) sponge and GnRH analogues (lecirelin or buserelin). All heifers received oestradiol benzoate plus one of the devices on Day 0 and PGF on Day 7 pm (device withdrawal). Heifers were detected in oestrus 36 h after PGF and inseminated 8-12 h later, while the remainder received GnRH 48 h after PGF and were inseminated on Day 10 (60 h). The number of heifers detected in oestrus on Day 8 and conception rate to AI on Day 9 were higher (p < 0.01) in the used-DIB than in the CIDR or MAP groups, while the opposite occurred with the pregnancy rate to FTAI on Day 10 (p < 0.01). There was no effect of progesterone source, GnRH analogue or their interaction on overall pregnancy rates (64.9%). Progesterone treatment of heifers during an Ovsynch protocol resulted in a larger pre-ovulatory follicle in beef heifers. Progesterone content of intravaginal devices in synchronization protocols is important for the timing of AI, as the use of low-progesterone devices can shorten the interval to oestrus.

Where there's smoke...carbon monoxide exposures in smoking and smoke-free workplaces.

Comparisons of expired air carbon monoxide levels in non-smoking staff in six licensed clubs (a smoking workplace) were made with those of non-smoking staff of a large public hospital (a smoke-free workplace). There was a significant difference between clubs and hospital in levels of CO at end of work. The average concentration of club workers was 8.7 ppm (Hospital, 5.3 ppm). Approximately one third of these non-smokers in licensed clubs exceeded 10 ppm putting them in the 'light smoker' category according to the manufacturer of the monitoring equipment. Club workers increased their CO level during work time by four times the increase of Hospital staff. This study suggests that there are significant gains to be made in reduction of intake of harmful passive-smoking products by removing tobacco smoke from workplaces.